Salt Form of a Multi-Arm Polymer-Drug Conjugate

ABSTRACT

Among other aspects, provided herein is a hydrohalide salt of a multi-arm water-soluble polyethylene glycol-drug conjugate, along with related methods of making and using the same. The hydrohalide salt is stably formed, and appears to be more resistant to hydrolytic degradation than the corresponding free base form of the conjugate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to each of U.S. Provisional Patent Application Ser. No. 61/262,463,filed 18 Nov. 2009, and U.S. Provisional Patent Application Ser. No.61/290,072, filed 24 Dec. 2009, both of which are incorporated herein byreference in their entireties.

FIELD

This disclosure relates generally to salt forms of water-solublepolymer-drug conjugates, pharmaceutical compositions thereof, andmethods for preparing, formulating, administering and using such mixedacid salt compositions. This disclosure also relates generally toalkoxylation methods for preparing alkoxylated polymeric materials froma previously isolated alkoxylated oligomer, as well as to compositionscomprising the alkoxylated polymeric material, methods for using thealkoxylated polymeric material, and the like.

BACKGROUND

Over the years, numerous methods have been proposed for improving thestability and delivery of biologically active agents. Challengesassociated with the formulation and delivery of pharmaceutical agentscan include poor aqueous solubility of the pharmaceutical agent,toxicity, low bioavailability, instability, and rapid in-vivodegradation, to name just a few. Although many approaches have beendevised for improving the delivery of pharmaceutical agents, no singleapproach is without its drawbacks. For instance, commonly employed drugdelivery approaches aimed at solving or at least ameliorating one ormore of these problems include drug encapsulation, such as in aliposome, polymer matrix, or unimolecular micelle, covalent attachmentto a water-soluble polymer such as polyethylene glycol, use of genetargeting agents, formation of salts, and the like.

Covalent attachment of a water-soluble polymer can improve thewater-solubility of an active agent as well as alter its pharmacologicalproperties. Certain exemplary polymer conjugates are described in U.S.Pat. No. 7,744,861, among others. In another approach, an active agenthaving acidic or basic functionalities can be reacted with a suitablebase or acid and marketed in salt form. Over half of all activemolecules are marketed as salts (Polymorphism in the PharmaceuticalIndustry, Hilfiker, R., ed., Wiley-VCH, 2006). Challenges with saltforms include finding an optimal salt, as well as controlling solidstate behavior during processing. Biopharmaceutical salts can beamorphous, crystalline, and exist as hydrates, solvents, variouspolymorphs, etc. Interestingly, rarely, if ever, are salt forms, letalone mixed acid salt forms, of polymer conjugates used in drugformulations.

Another challenge associated with preparing active agent conjugates ofwater-soluble polymers waters is the ability to prepare relatively purewater-soluble polymers in a consistent and reproducible method. Forexample, poly(ethylene glycol) (PEG) derivatives activated with reactivefunctional groups are useful for coupling to active agents (such assmall molecules and proteins), thereby forming a conjugate between thePEG and the active agent. When an active agent is conjugated to apolymer of poly(ethylene glycol) or “PEG,” the conjugated active agentis conventionally referred to as having been “PEGylated.”

When compared to the safety and efficacy of the active agent in theunconjugated form, the conjugated version exhibits different, and oftenclinically beneficial, properties. The commercial success of PEGylatedactive agents such as PEGASYS® PEGylated interferon alpha-2a(Hoffmann-La Roche, Nutley, N.J.), PEG-INTRON® PEGylated interferonalpha-2b (Schering Corp., Kennilworth, N.J.), and NEULASTA®PEG-filgrastim (Amgen Inc., Thousand Oaks, Calif.) demonstrates thedegree to which PEGylation has the potential to improve one or moreproperties of an active agent.

In preparing a conjugate, a polymeric reagent is typically employed toallow for a relatively straightforward synthetic approach for conjugatesynthesis. By combining a composition comprising a polymeric reagentwith a composition comprising the active agent, it is possible—under theappropriate reaction conditions—to carry out a relatively convenientconjugate synthesis.

The preparation of the polymeric reagent suitable to the regulatoryrequirements for drug products, however, is often challenging.Conventional polymerization approaches result in relatively impurecompositions and/or low yield. Although such impurities and yields maynot be problematic outside the pharmaceutical field, safety and costrepresent important concerns in the context of medicines for human use.Thus, conventional polymerization approaches are not suited for thesynthesis of polymeric reagents intended for the manufacture ofpharmaceutical conjugates.

There is a need in the art for alternative methods for preparingpolymeric reagents, particularly high molecular weight polymers, inrelatively high yield and purity. In the case of multiarm polymers,there is a dearth of available, desirable water soluble polymers thathave well controlled and well defined properties with the absence ofsignificant amounts of undesirable impurities. Thus one can readilyobtain for example a high molecular weight multi-arm poly(ethyleneglycol) but drug conjugates manufactured from commercial polymers canhave significant amounts (i.e. >8%) of polymer-drug conjugate havingeither very low or very high molecular weight biologically activeimpurities. This extent of active impurities in a drug composition maybe unacceptable and thus can render approval of such drugs challengingif not impossible.

SUMMARY

In one or more embodiments of the invention, the present disclosureprovides a hydrohalide salt form of a polymer-active agent conjugatecorresponding to structure (I):

where n is an integer ranging from about 20 to about 600, or from about20 to 500 (e.g., 40 to about 500) and, in terms of a compositioncomprising the above conjugate, greater than 95 mole percent (and insome instances greater than 96 mole percent, greater than 97 molepercent, and even greater than 98 mole percent) of basic nitrogens ofthe irinotecan portions of all the conjugates contained in thecomposition are protonated in hydrohalide (HX) salt form, wherein X isselected from fluoride, chloride, bromide, and iodide.

In one or more embodiments of the invention, the hydrohalide salt is ahydrochloride salt.

In one or more embodiments of the invention, n of a repeating monomer isan integer ranging from about 80 to about 150.

In one or more embodiments of the invention, n for any instance of(OCH₂CH₂)_(n) is of about 113.

In one or more embodiments of the invention, the hydrohalide salt is ahydrochloride salt and the weight average molecular weight of theconjugate is about 23,000 daltons.

In one or more embodiments of the invention, a method for preparing ahydrohalide salt of a water-soluble polymer-active agent conjugate [suchas the water-soluble polymer-active agent of conjugate of structure (1)]is provided], the method comprising the steps of: (i) treating aglycine-irinotecan hydrohalide in protected form (II),

with a molar excess of hydrohalic acid to thereby remove the protectinggroup to form glycine-irinotecan hydrohalide,

(ii) coupling the deprotected glycine-irinotecan hydrohalide from step(i) with a 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-succinimide,

in the presence of a base to form 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt (also referredto as pentaerythritolyl-4-arm-(PEG-1-methylene-2-oxo-vinylamino acetatelinked-irinotecan hydrohalide salt)),

(iii) recovering the 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt byprecipitation. With respect to this method, the polymer reagent used tocarry out the method is not particularly limited and the other polymerreagents bearing an activated ester can be substituted for the4-arm-pentaerythritolyl-polyethylene glycol-carboxymethyl-succinimide.

In one or more embodiments of the invention, a recovered4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide is contained withina composition in which greater than 95 mole percent (and in someinstances greater than 96 mole percent, greater than 97 mole percent,and even greater than 98 mole percent) of basic nitrogens of theirinotecan portions of all the conjugates contained in the compositionare protonated in hydrohalide (HX) salt form, wherein X is selected fromfluoride, chloride, bromide, and iodide.

In one or more embodiments of the invention, the glycine-irinotecanhydrohalide in protected form is treated with a ten-fold or greatermolar excess of hydrohalic acid to thereby remove the protecting groupto form glycine-irinotecan hydrohalide.

In one or more embodiments of the invention, the glycine-irinotecanhydrohalide in protected form is treated with a molar excess ofhydrohalic acid in a range of ten-fold to 25-fold to thereby remove theprotecting group to form glycine-irinotecan hydrohalide.

In one or more embodiments of the invention, the glycine-irinotecanhydrohalide in protected form istert-butyloxycarbonyl(Boc)-glycine-irinotecan hydrochloride, wherein theamino group of glycine is Boc-protected.

In one or more embodiments of the invention, the glycine-irinotecanhydrohalide in step (i) is glycine-irinotecan hydrochloride in protectedform, and the glycine-irinotecan hydrochloride in protected form istreated with hydrochloric acid to remove the protecting group.

In one or more embodiments of the invention, the glycine-irinotecanhydrochloride in protected form is treated with a solution ofhydrochloric acid in dioxane.

In one or more embodiments of the invention, a method for preparing ahydrohalide salt of a water-soluble polymer-active agent conjugatemethod further comprises isolating the glycine-irinotecan hydrohalide(e.g., by precipitation, by addition of methyltertbutylether, “MTBE”)prior to step the coupling step with a polymer reagent.

In one or more embodiments of the invention, the base used in thecoupling step is an amine (e.g., trimethylamine, triethylamine, anddimethylamino-pyridine).

In one or more embodiments of the invention, the coupling step iscarried out in a chlorinated solvent.

In one or more embodiments of the invention, the step of recovering the4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt comprisesaddition of methyltertbutyl ether.

In one or more embodiments of the invention, the method for preparing ahydrohalide salt of a water-soluble polymer-active agent conjugatefurther comprises the step of (iv) analyzing (e.g., by ionchromatography) the recovered 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt for halidecontent, and, in the event the halide content is less than 95 molepercent, (v) dissolving the recovered4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt in ethylacetate, and adding additional hydrohalic acid to thereby form the4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt having a halidecontent of greater than 95 mole percent.

In one or more embodiments of the invention, the hydrohalic acid addedis in the form of an ethanol solution.

In one or more embodiments of the invention, in a method in which thestep of adding additional hydrohalic acid is carried out, the methodfurther comprises recovering the 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide by precipitation(which may be effected by, for example, cooling).

In one or more embodiments of the invention, a hydrohalide salt of4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan is provided by carrying outmethods described herein.

In one or more embodiments of the invention, a pharmaceuticallyacceptable composition is provided, the composition comprising ahydrohalide salt (e.g., hydrochloride salt) of the compoundcorresponding to structure (I), 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan, and a pharmaceuticallyacceptable excipient.

In one or more embodiments of the invention, a composition is provided,the composition comprising a hydrochloride salt according to any one ormore of the embodiments described herein, and (ii) lactate buffer, inlyophilized form. In one or more embodiments of the invention, thepharmaceutically acceptable composition is a sterile composition. In oneor more embodiments of the invention, the pharmaceutically acceptablecomposition is optionally provided in a container (e.g., vial),optionally containing the equivalent of a 100-mg dose of irinotecan.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a conjugate-containing compositiondescribed herein to an individual suffering from one or more types ofcancerous solid tumors, wherein the conjugate-containing composition isoptionally dissolved in a solution of 5% w/w dextrose. In one or moreembodiments of the invention, administration is effected via intravenousinfusion.

In one or more embodiments of the invention, a method of treating amammal suffering from cancer is provided, the method comprisingadministering a therapeutically effective amount of a hydrohalide salt(such as a hydrochloride salt) of 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan. The hydrohalide salt isadministered to the mammal effective to produce a slowing or inhibitionof solid tumor growth in the subject. In one or more embodiments of theinvention, the cancerous solid tumor is selected from the groupconsisting of colorectal, ovarian, cervical, breast and non-small celllung.

In one or more embodiments of the invention, a4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt is provided,wherein the salt is an anti-cancer agent for the manufacture of amedicament for treating cancer.

In one or more embodiments of the invention, a composition is provided,the composition comprising an alkoxylated polymeric product prepared bya method comprising the step of alkoxylating in a suitable solvent apreviously isolated alkoxylatable oligomer to form an alkoxylatedpolymeric product, wherein the previously isolated alkoxylatableoligomer has a known and defined weight-average molecular weight ofgreater than 300 Daltons (e.g., greater than 500 Daltons).

In one or more embodiments of the invention, a composition is provided,the composition comprising an alkoxylated polymeric product having apurity of greater than 92 wt % and the total combined content of highmolecular weight products and diols is less than 8 wt % (e.g., less than2 wt %), as determined by, for example, gel filtration chromatography(GFC) analysis.

In one or more embodiments of the invention, the alkoxylated polymerproduct has the following structure:

wherein each n is an integer from 20 to 1000 (e.g., from 50 to 1000).

In one or more embodiments of the invention, a method is provided, themethod comprising the steps of (i) alkoxylating in a suitable solvent apreviously isolated alkoxylatable oligomer to form an alkoxylatedpolymeric material, wherein the previously isolated alkoxylatableoligomer has a known and defined weight-average molecular weight ofgreater than 300 Daltons (e.g., greater than 500 Daltons), and (ii)optionally, further activating the alkoxylated polymeric product toprovide an activated alkoxylated polymeric product that is useful as(among other things) a polymeric reagent for preparing polymer-drugconjugates.

In one or more embodiments of the invention, a method is provided, themethod comprising the step of activating an alkoxylated polymericproduct obtained from and/or contained within a composition comprisingan alkoxylated polymeric product having a purity of greater than 90% tothereby form an activated alkoxylated polymeric product that is usefulas (among other things) a polymer reagent for preparing polymer-drugconjugates.

In one or more embodiments of the invention, a method is provided, themethod comprising the step of conjugating an activated alkoxylatedpolymeric product to an active agent, wherein the activated alkoxylatedpolymeric product was prepared by a method comprising the step ofactivating an alkoxylated polymeric product obtained from and/orcontained within a composition comprising an alkoxylated polymericproduct having a purity of greater than 90% to thereby form an activatedalkoxylated polymeric product.

In one or more embodiments of the invention, a mixed salt of awater-soluble polymer-active agent conjugate is provided, the conjugatehaving been prepared by coupling (under conjugation conditions) anamine-bearing active agent (e.g., a deprotected glycine-irinotecan) to apolymer reagent (e.g., a 4-arm pentaerythritolyl-poly(ethyleneglycol)-carboxymethyl succinimide) in the presence of a base to form aconjugate, wherein the conjugate is in the form of a mixed saltconjugate (e.g., the conjugate possesses nitrogen atoms, each one ofwhich will either be protonated or unprotonated, where any givenprotonated amino group is an acid salt possessing one of two differentanions), and further wherein, optionally, the polymer reagent isprepared from an alkoxylation product prepared as described herein.

Additional embodiments of the present method, compositions, and the likewill be apparent from the following description, drawings, examples, andclaims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the present invention. Additional aspects and advantagesof the present invention are set forth in the following description andclaims, particularly when considered in conjunction with theaccompanying examples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the results of accelerated stressstability studies on three different samples of“4-arm-PEG-Gly-Irino-20K” (corresponding to4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan), each having a differentcomposition with respect to relative amounts of trifluoroacetic acid(TFA) and hydrochloride salts, as well as free base. Samples testedincluded >99% HCl salt (<1% free base, triangles), 94% total salt (6%free base, squares), and 52% total salt (48% free base, circles). Thesamples were stored at 25° C. and 60% relative humidity; the plotillustrates degradation of compound over time as described in detail inExample 3.

FIG. 2 is a graph illustrating the increase in free irinotecan over timein samples of 4-arm-PEG-Gly-Irino-20K stored at 40° C. and 75% relativehumidity, each having a different composition with respect to relativeamounts of trifluoroacetic acid and hydrochloride salts, as well as freebase. Samples tested correspond to product containing >99% HCl salt (<1%free base, squares) and product containing 86% total salts (14% freebase, diamonds), as described in Example 3.

FIG. 3 is a graph illustrating the increase over time in small PEGspecies (PEG degradation products) in samples of4-arm-PEG-Gly-Irinio-20K stored at 40° C. and 75% relative humidity, asdescribed in detail in Example 3. Samples tested correspond to productcontaining >99% HCl salt (<1% free base, squares) and product containing86% total salts (14% free base, diamonds).

FIG. 4 is a compilation of overlays of chromatograms exhibiting releaseof irinotecan via hydrolysis from mono-(DS-1), di-(DS-2), tri-(DS-3) andtetra-irinotecan substituted (DS-4) 4-arm-PEG-Gly-Irino-20K as describedin detail in Example 5.

FIG. 5 is a graph illustrating the results of hydrolysis of variousspecies of 4-arm-PEG-Gly-Irino-20K as described above in aqueous bufferat pH 8.4 in the presence of porcine carboxypeptidase B in comparison tohydrolysis kinetics modeling data as described in Example 5. For thekinetics model, the hydrolysis of all species was assumed to be 1^(st)order kinetics. The 1^(st) order reaction rate constant fordisappearance of DS4 (0.36 hr⁻¹) was used to generate all curves.

FIG. 6 is a graph illustrating the hydrolysis of various species of4-arm-PEG-Gly-Irino-20K as described above in human plasma in comparisonto hydrolysis kinetics modeling data. Details are provided in Example 5.For the kinetics model, the hydrolysis of all species was assumed to be1^(st) order kinetics. The 1^(st) order reaction rate constant fordisappearance of DS 4 (0.26 hr⁻¹) was used to generate all curves.

FIG. 7 is a chromatogram following gel filtration chromatography of amaterial prepared a described in Example 7.

FIG. 8 is a chromatogram following gel filtration chromatography of amaterial prepared a described in Example 8.

DETAILED DESCRIPTION

Various aspects of the invention now will be described more fullyhereinafter. Such aspects may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties. In the event of an inconsistency between the teachings ofthis specification and the art incorporated by reference, the meaning ofthe teachings in this specification shall prevail.

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

A “functional group” is a group that may be used, under normalconditions of organic synthesis, to form a covalent linkage between theentity to which it is attached and another entity, which typically bearsa further functional group. The functional group generally includesmultiple bond(s) and/or heteroatom(s). Preferred functional groups aredescribed herein.

The term “reactive” refers to a functional group that reacts readily orat a practical rate under conventional conditions of organic synthesis.This is in contrast to those groups that either do not react or requirestrong catalysts or impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups thatmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andin P. J. Kocienski, Protecting Groups, Third Ed. Thieme Chemistry, 2003,and references cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) ranges from 3 to about 3000,and the terminal groups and architecture of the overall PEG may vary.

A water-soluble polymer may bear one or more “end-capping group,” (inwhich case it can stated that the water-soluble polymer is “end-capped.”With regard to end-capping groups, exemplary end-capping groups aregenerally carbon- and hydrogen-containing groups, typically comprised of1-20 carbon atoms and an oxygen atom that is covalently bonded to thegroup. In this regard, the group is typically alkoxy (e.g., methoxy,ethoxy and benzyloxy) and with respect to the carbon-containing groupcan optionally be saturated or unsaturated, as well as aryl, heteroaryl,cyclo, heterocyclo, and substituted forms of any of the foregoing.

The end-capping group can also comprise a detectable label. When thepolymer has an end-capping group comprising a detectable label, theamount or location of the polymer and/or the moiety (e.g., active agent)to which the polymer is attached can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that is atleast 35% (by weight), preferably greater than 70% (by weight), and morepreferably greater than 95% (by weight) soluble in water at roomtemperature. Typically, a water-soluble polymer or segment will transmitat least about 75%, more preferably at least about 95% of light,transmitted by the same solution after filtering.

The term “activated,” when used in conjugation with a particularfunctional group, refers to a reactive functional group that reactsreadily with an electrophile or nucleophile on another molecule. This isin contrast to those groups that require strong bases or highlyimpractical reaction conditions in order to react (i.e., a “nonreactive”or “inert” group).

“Electrophile” refers to an ion or atom or a neutral or ionic collectionof atoms having an electrophilic center, i.e. a center that is electronseeking or capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or a neutral or ionic collectionof atoms having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or capable of reacting with an electrophile.

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive group beingprotected as well as the reaction conditions to be employed and thepresence of additional reactive or protecting groups in the molecule, ifany. Protecting groups known in the art can be found in Greene, T. W.,et al. PROTECTIVE GROUPS IN ORGANIC SYNTHFSIS, 3rd ed., John Wiley &Sons, New York, N.Y. (1999).

“Molecular mass” in the context of a water-soluble polymer such as PEG,refers to the weight average molecular weight of a polymer, typicallydetermined by size exclusion chromatography, light scatteringtechniques, or intrinsic viscosity determination in an organic solventlike 1,2,4-trichlorobenzene.

The terms “spacer” and “spacer moiety” are used herein to refer to anatom or a collection of atoms optionally used to link interconnectingmoieties such as a terminus of a series of monomers and an electrophile.The spacer moieties of the invention may be hydrolytically stable or mayinclude a physiologically hydrolyzable or enzymatically degradablelinkage.

A “hydrolyzable” bond is a relatively labile bond that reacts with water(i.e., is hydrolyzed) under physiological conditions. The tendency of abond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Illustrative hydrolytically unstablelinkages include carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond thatis substantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, and the like.Generally, a hydrolytically stable linkage is one that exhibits a rateof hydrolysis of less than about 1-2% per day under physiologicalconditions. Hydrolysis rates of representative chemical bonds can befound in most standard chemistry textbooks.

“Multi-armed” in reference to the geometry or overall structure of apolymer refers to polymer having 3 or more polymer-containing “arms”connected to a “core” molecule or structure. Thus, a multi-armed polymermay possess 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymerarms, 7 polymer arms, 8 polymer arms or more, depending upon itsconfiguration and core structure. One particular type of multi-armedpolymer is a highly branched polymer referred to as a dendritic polymeror hyperbranched polymer having an initiator core of at least 3branches, an interior branching multiplicity or 2 or greater, ageneration of 2 or greater, and at least 25 surface groups within asingle dendrimer molecule. For the purposes herein, a dendrimer isconsidered to possess a structure distinct from that of a multi-armedpolymer. That is to say, a multi-armed polymer as referred to hereinexplicitly excludes dendrimers. Additionally, a multi-armed polymer asprovided herein possesses a non-crosslinked core.

A “dendrimer” or “hyperbranched polymer” is a globular, sizemonodisperse polymer in which all bonds emerge radially from a centralfocal point or core with a regular branching pattern and with repeatunits that each contribute a branch point. Dendrimers are typicallyalthough not necessarily formed using a nano-scale, multistepfabrication process. Each step results in a new “generation” that hastwo or more times the complexity of the previous generation. Dendrimersexhibit certain dendritic state properties such as core encapsulation,making them unique from other types of polymers.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms. A multi-arm polymer may have onebranch point or multiple branch points, so long as the branches are notregular repeats resulting in a dendrimer.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

“Alkyl” refers to a hydrocarbon chain ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, isopropyl, n-butyl, n-pentyl, 2-methyl-1-butyl,3-pentyl, 3-methyl-3-pentyl, and the like.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl and t-butyl.

“Cycloalkyl” refers to a saturated cyclic hydrocarbon chain, includingbridged, fused, or spiro cyclic compounds, preferably made up of 3 toabout 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl.” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

As used herein, “alkenyl” refers to branched and unbranched hydrocarbongroups of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl (vinyl), 2-propen-1-yl (allyl), isopropenyl,3-buten-1-yl, and the like.

The term “alkynyl” as used herein refers to branched and unbranchedhydrocarbon groups of 2 to 15 atoms in length, containing at least onetriple bond, such as ethynyl, 1-propynyl, 3-butyn-1-yl, 1-octyn-1-yl,and so forth.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrecenyl,naphthacenyl, and the like.

“Substituted phenyl” and “substituted aryl” denote a phenyl group andaryl group, respectively, substituted with one, two, three, four, orfive (e.g., 1-2, 1-3, 1-4, or 1-5 substituents) chosen from halo (F, Cl,Br, I), hydroxyl, cyano, nitro, alkyl (e.g., C₁₋₆ alkyl), alkoxy (e.g.,C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, and so forth.

An inorganic acid is an acid that is absent carbon atoms. Examplesinclude hydrohalic acids, nitric acid, sulfuric acid, phosphoric acidand the like.

“Hydrohalic acid” means a hydrogen halide such as hydrofluoric acid(HF), hydrochloric acid (HCl), hydrobromic acid (HBr), and hydroiodicacid (HI).

“Organic acid” means any organic compound (i.e., having at least onecarbon atom) possessing one or more carboxy groups (—COOH). Somespecific examples include formic acid, lactic acid, benzoic acid, aceticacid, trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid,mixed chlorofluoroacetic acids, citric acid, oxalic acid, and the like.

“Active agent” as used herein includes any agent, drug, compound, andthe like which provides some pharmacologic, often beneficial, effectthat can be demonstrated in-vivo or in vitro. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient. Asused herein, especially in reference to synthetic approaches describedherein, a “active agent” is meant to encompass derivatized or linkermodified versions thereof, such that upon administration in vivo, theparent “bioactive” molecule is released.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to an excipient that can be included in a compositioncomprising an active agent and that causes no significant adversetoxicological effects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of an active agent present in a pharmaceuticalpreparation that is needed to provide a desired level of active agentand/or conjugate in the bloodstream or in a target tissue or site in thebody. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofthe pharmaceutical preparation, intended patient population, and patientconsiderations, and can readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

“Multi-functional” in the context of a polymer means a polymer having 3or more functional groups, where the functional groups may be the sameor different, and are typically present on the polymer termini.Multi-functional polymers will typically contain from about 3-100functional groups, or from 3-50 functional groups, or from 3-25functional groups, or from 3-15 functional groups, or from 3 to 10functional groups, i.e., contains 3, 4, 5, 6, 7, 8, 9 or 10 functionalgroups.

“Difunctional” and “bifunctional” are used interchangeably herein andmean an entity such as a polymer having two functional groups containedtherein, typically at the polymer termini. When the functional groupsare the same, the entity is said to be homodifunctional orhomobifunctional. When the functional groups are different, the entityis said to be heterodifunctional or heterobifunctional.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The terms “subject,” “individual” and “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals and pets. Such subjects are typically suffering from orprone to a condition that can be prevented or treated by administrationof a water-soluble polymer-active agent conjugate as described herein.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Treatment” and “treating” of a particular condition include: (1)preventing such a condition, i.e. causing the condition not to develop,or to occur with less intensity or to a lesser degree in a subject thatmay be exposed to or predisposed to the condition but does not yetexperience or display the condition, and (2) inhibiting the condition,i.e., arresting the development or reversing the condition.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

A “small molecule” is an organic, inorganic, or organometallic compoundtypically having a molecular weight of less than about 1000, preferablyless than about 800 daltons. Small molecules as referred to hereinencompass oligopeptides and other biomolecules having a molecular weightof less than about 1000.

A “peptide” is a molecule composed of from about 13 to 50 or so aminoacids. An oligopeptide typically contains from about 2 to 12 aminoacids.

Unless explicitly stated to the contrary, the terms “partial mixed salt”and “mixed salt” as used herein are used interchangeably, and, in thecase of a polymer conjugate (and corresponding compositions comprising aplurality of such polymer conjugates), refer to a conjugates andcompositions comprising one or more basic amino (or other basic nitrogencontaining) groups, where (i) any given one of the basic amino groups inthe conjugate or conjugate composition is either non-protonated orprotonated and (ii) with respect to any given protonated basic aminogroup, the protonted basic amino group will have one of two differentcounterions. (The term “partial mixed salt” refers to the feature wherenot all amino groups in the compound or composition are protonated—hencethe composition being a “partial” salt, while “mixed” refers to thefeature of multiple counterions). A mixed salt as provided hereinencompasses hydrates, solvates, amorphous forms, crystalline forms,polymorphs, isomers, and the like.

An amine (or other basic nitrogen) group that is in “free base” form isone where the amine group, i.e., a primary, secondary, or tertiaryamine, possesses a free electron pair. The amine is neutral, i.e., isuncharged.

An amine group that is in “protonated form” exists as a protonatedamine, so that the amino group is positively charged. As used herein, anamine group that is protonated can also be in the form of an acidaddition salt resulting from reaction of the amine with an acid such asan inorganic acid or an organic acid.

The “mole percent” of an active agent's amino groups refers to thefraction or percentage of amino groups in an active agent moleculecontained in a polymer conjugate that are in one particular form oranother, where the total mole percent of amino groups in the conjugateis 100 percent.

As used herein, “psi” means pounds per square inch.

Overview Hydrohalide Salts, Alkoxylation Methods, and Compositions ofConjugates (and Hydrohalide Salt Forms Thereof) Prepared from PolymerReagents Prepared from Polymeric Products Prepared from the AlkoxylationMethods

Hydrohalide Salts:

As previously indicated, in one or more embodiments of the invention, awater-soluble polymer and active agent conjugate is provided, whereinthe conjugate is in the form of a hydrohalide salt (e.g., a hydrohalidesalt of 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan). Such conjugates representnovel solid state forms. A process to reproducibly prepare an irinotecanconjugate-containing composition is provided, wherein—with respect toall of the irinotecan conjugates in the composition—greater than 95 molepercent of all of irinotecan's basic nitrogen atoms are protonated in ahydrohalide (HX) salt form has been discovered and is provided herein.It has further been discovered that the hydrohalide salt demonstratesenhanced stability towards hydrolytic degradation, e.g., when comparedto the free base form of the conjugate. See, e.g., Example 3.

By way of background, during the preparation of the4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan, as described in detail inExample 1, it was discovered that, in spite of treatment with base, theproduct was generally formed as a mixed acid salt having irinotecan'sbasic nitrogen atoms, e.g., amino groups, in either protonated orunprotonated form, where any given protonated amino group was an acidsalt possessing one of two different anions (e.g., trifluoroacetate orchloride). In an attempt to further explore the resulting composition, amethod for preparing substantially pure hydrohalide salt was devised. Asdescribed generally above, the hydrohalide salt described hereinpossesses certain notable and advantageous properties. The structuralcharacteristics, properties, method of making and using, and additionalfeatures of the 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt, among otherfeatures, are described herein.

Briefly, the features of a hydrohalide salt, e.g., hydrochloride salt,of a water-soluble polymer-active agent conjugate typically include thefollowing. Generally speaking, the compound is a conjugate ofmulti-armed poly(ethylene glycol) polymer and irinotecan. Irinotecan, asis evident from its structure, possesses one or more basic amine groups(or other basic nitrogen atoms), i.e., having a pK from about 7.5 toabout 11.5 after conjugation to the multi-armed polymer core (i.e., theactive agent possesses one or more basic amine or other nitrogencontaining groups after conjugation to the water soluble polymer). Theresulting conjugate is a hydrohalide salt, i.e., where the basicnitrogen atoms are protonated as a hydrohalide salt (HX, where X isselected from fluoride, chloride, bromide and iodide).

As used herein, a hydrohalide salt having greater than 95 mole percentof irinotecan's basic nitrogen atoms protonated as the hydrohaliderefers to the “bulk product” rather than necessarily referring toindividual molecular species contained within the bulk product. Thus,individual molecular species contained within the salt, due to thenumber of polymer arms within the conjugate structure, may contain asmall number of amine groups that are in free base form as well as inprotonated form as described above. Moreover, the 4-armPEG-carboxymethyl conjugate core, in general, may be less than fullysubstituted with covalently attached irinotecan, this feature to bedescribed in greater detail below.

Alkoxylation Methods:

As also previously indicated, in one or more embodiments of theinvention, a method is provided, the method comprising the step ofalkoxylating in a suitable solvent a previously isolated alkoxylatableoligomer to form an alkoxylated polymeric product, wherein thepreviously isolated alkoxylatable oligomer has a known and definedweight-average molecular weight of greater than 300 Daltons (e.g.,greater than 500 Daltons). Among other advantages, the alkoxylationmethods provided herein result in polymeric products that are superior(e.g., in terms of consistency and purity) than polymeric productsprepared by previously known methods.

Compositions of Conjugates (and Hydrohalide Salt Forms Thereof) Preparedfrom Polymer Reagents Prepared from Polymeric Products Prepared from theAlkoxylation Methods:

As also previously indicated, in one or more embodiments of theinvention, a hydrohalide salt of a water-soluble polymer-active agentconjugate is provided, wherein the conjugate is prepared by coupling(under conjugation conditions) an amine-bearing active agent (e.g., adeprotected glycine-irinotecan) to a polymer reagent (e.g., 4-armpentaerythritolyl-poly(ethylene glycol)-carboxymethyl succinimide) inthe presence of a base to form a conjugate, wherein the conjugate is ahydrohalide salt conjugate (e.g. the conjugate possesses nitrogen atoms,each one of which will either be protonated or unprotonated, where anygiven protonated amino group is a hydrohalide salt), and furtherwherein, optionally, the polymer reagent is prepared from a alkoxylationproduct prepared as described herein.

Conjugates The Polymer Generally

Water-soluble polymer-active agent conjugates (regardless of thespecific form taken, e.g., a base form, salt form, mixed salt, and soforth) include a water-soluble polymer. Typically, in order to form aconjugate, a water-soluble polymer—in the form of a polymer reagent—iscoupled (under conjugation conditions) to an active agent at anelectrophile or nucleophile contained within the active agent. Forexample, a water-soluble polymer (again, in the form of a polymerreagent bearing, e.g., an activated ester) can be coupled to an activeagent possessing one or more basic amine groups (or other basic nitrogenatoms), i.e., an amine having a pK from about 7.5 to about 11.5(determined after conjugation).

The water-soluble polymer component of the conjugate is typically awater-soluble and non-peptidic polymer. Representative polymers includepoly(alkylene glycol), poly(oletinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharide), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), orcopolymers or terpolymers thereof. One particular water-soluble polymeris polyethylene glycol or PEG comprising the repeat unit (CH₂CH₂O)_(n)—,where n ranges from about 3 to about 2700 or even greater, or preferablyfrom about 25 to about 1300. Typically, the weight average molecularweight of the water-soluble polymer in the conjugate ranges from about100 daltons to about 150,000 daltons. Illustrative overall molecularweights for the conjugate may range from about 800 to about 80,000daltons, or from about 900 to about 70,000 daltons. Additionalrepresentative molecular weight ranges are from about 1,000 to about40,000 daltons, or from about 5.000 to about 30,000 daltons, or fromabout 7500 daltons to about 25,000 daltons, or even from about 20,000 toabout 80,000 daltons for higher molecular weight embodiments of theinstant salts.

The water-soluble polymer can be in any of a number of geometries orforms, including linear, branched, forked. In exemplary embodiments, thepolymer is often linear or multi-armed. Water-soluble polymers can beobtained commercially as simply the water-soluble polymer. In addition,water-soluble polymers can be conveniently obtained in an activated formas a polymer reagent (which optionally may be coupled to an active agentwithout further modification or activation). Descriptions ofwater-soluble polymers and polymer reagents can be found in NektarAdvanced PEGylation Catalog, 2005-2006, “Polyethylene Glycol andDerivatives for Advanced PEGylation” and are available for purchase fromNOF Corporation and JenKem Technology USA, among others.

An exemplary branched polymer having two polymer arms in a branchedpattern is the following, often referred to as PEG-2 or mPEG-2:

wherein

indicates the location for additional atoms to form any of functionalgroups suitable for reaction with an electrophile or nucleophilecontained within an active agent. Exemplary functional groups includeNHS ester, aldehyde, and so forth.

For polymer structures described herein that contain the variable, “n,”such variable corresponds to an integer and represents the number ofmonomer subunits within the repeating monomeric structure of thepolymer.

On exemplary architecture for use in preparing the conjugates aremulti-arm water-soluble polymer reagents having for example 3, 4, 5, 6or 8 polymer arms, each optimally bearing a functional group. Amulti-arm polymer reagent may possess any of a number of cores (e.g., apolyol core) from which the polymer arms emanate. Exemplary polyol coresinclude glycerol, glycerol dimer(3,3′-oxydipropane-1,2-diol)trimethylolpropane, sugars (such as sorbitolor pentaerythritol, pentaerythritol dimer), and glycerol oligomers, suchas hexaglycerol or3-(2-hydroxy-3-(2-hydroxyethoxy)propoxy)propane-1,2-diol, and otherglycerol condensation products. Exemplary, the cores and the polymerarms emanating therefrom can be of the following formulae:

In an exemplified embodiment, the water soluble polymer is a 4-armpolymer as shown above, where n may range from about 20 to about 500, orfrom about 40 to about 500.

In the multi-arm embodiments described herein, each polymer armtypically has a molecular weight corresponding to one of the following:200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500,18,000, 19.000, 20,000 Daltons or greater. Overall molecular weights forthe multi-armed polymer configurations described herein (that is to say,the molecular weight of the multi-armed polymer as a whole) generallycorrespond to one of the following: 800, 1000, 1200, 1600, 2000, 2400,2800, 3200, 3600, 4000, 5000, 6000, 8000, 10,000, 12,000, 15,000,16,000, 20,000, 24,000, 25,000, 28,000, 30.000, 32,000, 36,000, 40,000,45,000, 48,000, 50,000, 60,000, 80,000 or 100,000 or greater.

The water-soluble polymer, e.g., PEG, may be covalently linked to theactive agent via an intervening linker. The linker may contain anynumber of atoms. Generally speaking, the linker has an atom lengthsatisfying one or more of the following ranges: from about 1 atom toabout 50 atoms; from about 1 atom to about 25 atoms; from about 3 atomsto about 12 atoms; from about 6 atoms to about 12 atoms; and from about8 atoms to about 12 atoms. When considering atom chain length, onlyatoms contributing to the overall distance are considered. For example,a linker having the structure, —CH₂—C(O)—NH₁—CH₂ CH₂ O—CH₂ CH₂ O—C(O)—O—is considered to have a chain length of 11 atoms, since substituents arenot considered to contribute significantly to the length of the linker.Illustrative linkers include bifunctional compounds such as amino acids(e.g., alanine, glycine, isoleucine, leucine, phenylalanine, methionine,serine, cysteine, sarcosine, valine, lysine, and the like). The aminoacid may be a naturally-occurring amino acid or a non-naturallyoccurring amino acid. Suitable linkers also include oligopeptides.

The multi-arm structures above are drawn primarily to illustrate thepolymer core having PEG chains attached thereto, and although not drawnexplicitly, depending upon the nature of the active agent and attachmentchemistry employed, the final structure may optionally include anadditional ethylene group, —CH₂CH₂—, attached to the oxygen atoms at theterminus of each polymer arm, and/or may optionally contain any of anumber of intervening linker atoms to facilitate covalent attachment toan active agent. In a particular embodiment, each of the PEG armsillustrated above further comprises a carboxy methyl group, —CH₂—C(O)—,covalently attached to the terminal oxygen atom.

New Alkoxylation Method for Improved Polymer Compositions

As indicated previously, water-soluble polymers that have utility in(for example) preparing conjugates with active agents (as well as saltforms thereof) can be obtained commercially. As further describedherein, however, methods for preparing water-soluble polymers—whichmethods distinguish over previously described methods for preparingwater-soluble polymers—are provided that are particularly suited forpreparing conjugates with active agents (as well as salt forms thereof).

In this regard, a method is provided, the method comprising the step ofalkoxylating in a suitable solvent a previously isolated alkoxylatableoligomer to form an alkoxylated polymeric product, wherein thepreviously isolated alkoxylatable oligomer has a known and definedweight-average molecular weight of greater than 300 Daltons (e.g.,greater than 500 Daltons).

The Alkoxylatine Step in the New Alkoxylation Method

The alkoxylating step is carried out using alkoxylation conditions, suchthat the sequential addition of monomers is effected through repeatedreactions of an oxirane compound. When the alkoxylatable oligomerinitially has one or more hydroxyl functional groups, one or more ofthese hydroxyl groups in the alkoxylatable oligomer will be convertedinto a reactive alkoxide by reaction with a strong base. Then, anoxirane compound reacts with an alkoxylatable functional group (e.g., areactive alkoxide), thereby not only adding to the reactive alkoxide,but doing so in a way that also terminates in another reactive alkoxide.Thereafter, repeated reactions of an oxirane compound at the reactivealkoxide terminus of the previously added and reacted oxirane compoundeffectively produces a polymer chain.

Although each of the one or more alkoxylatable functional groups ispreferably hydroxyl, other groups such as amines, thiols and thehydroxyl group of a carboxylic acid can serve as an acceptablealkoxylatable functional group. Also, because of the acidity of thehydrogens of the alpha carbon atoms in aldehydes, ketones, nitriles andamides, addition at the alpha carbon atoms of these groups can serve asan acceptable alkoxylatable functional group.

The oxirane compound contains an oxirane group and has the followingformula:

wherein (with respect to this structure):

R¹ is selected from the group consisting of H and alkyl (preferablylower alkyl when alkyl);

R² is selected from the group consisting of H and alkyl (preferablylower alkyl when alkyl);

R³ is selected from the group consisting of H and alkyl (preferablylower alkyl when alkyl); and

R⁴ is selected from the group consisting of H and alkyl (preferablylower alkyl when alkyl).

With respect to the above oxirane compound formula, it is particularlypreferred that each of R¹, R², R³ and R⁴ is H, and it is preferred thatonly one of R¹, R², R³ and R⁴ is alkyl (e.g., methyl and ethyl) and theremaining substituents are H. Exemplary oxirane compounds are ethyleneoxide, propylene oxide and 1,2-butylene oxide. The amount of oxiranecompound added to result in optimal alkoxylation conditions depends upona number of factors, including the amount of starting alkoxylatablecoligomer, the desired size of the resulting alkoxylated polymericmaterial and the number of alkoxylatable functional groups on thealkoxylatable oligomer. Thus, when a larger alkoxylated polymericmaterial is desired, relatively more oxirane compound is present in thealkoxylation conditions. Similarly, if (Oa) represents the amount ofoxirane compound needed to achieve a given size of polymer “growth” on asingle alkoxylatable functional group, then an alkoxylatable oligomerbearing two alkoxylatable functional groups requires 2×(Oa), analkoxylatable oligomer bearing three alkoxylatable functional groupsrequires 3×(Oa), an alkoxylatable oligomer bearing four alkoxylatablefunctional groups requires 4×(Oa) and so on. In all cases, one ofordinary skill in the art can determine an appropriate amount of oxiranecompound required for alkoxylation conditions by taking into account thedesired molecular weight of alkoxylated polymeric material and followingroutine experimentation.

The alkoxylation conditions include the presence of a strong base. Thepurpose of the strong base is to deprotonate each acidic hydrogen (e.g.,the hydrogen of a hydroxyl group) present in the alkoxylatable oligomerand form an alkoxide ionic species (or an ionic species for non-hydroxylalkoxylatable functional groups). Preferred strong bases for use as partof the alkoxylation conditions are: alkali metals, such as metallicpotassium, metallic sodium, and alkali metals mixtures such assodium-potassium alloys: hydroxides, such as NaOH and KOH; and alkoxides(e.g., present following addition of an oxirane compound). Other strongbases can be used and can be identified by one of ordinary skill in theart. For example a given base can be used as a strong base herein if thestrong base can form an alkoxide ionic species (or an ionic species fornon-hydroxyl alkoxylatable functional groups) and also provide a cationthat does not encumber the alkoxide ionic species so as to hinder (oreffectively hinder through an impractically slow) reaction of thealkoxide ionic species with the oxirane molecule. The strong base ispresent in a generally small and calculated amount, which amount canfall into one or more of the following ranges: from 0.001 to 10.0 weightpercent based upon the weight of the total reaction mixture; and from0.01 to about 6.0 weight percent based upon the weight of the totalreaction mixture.

The alkoxylation conditions include a temperature suitable foralkoxylation to occur. Exemplary temperatures that may be suitable foralkoxylation to occur include those falling into one or more of thefollowing ranges: from 10° C. to 260° C.; from 20° C. to 240° C.; from30° C. to 220° C.; from 40° C. to 200° C.; from 50° C. to 200° C.; from80° C. to 140° C.; and from 100° C. to 120° C.

The alkoxylation conditions include a pressure suitable for alkoxylationto occur. Exemplary pressures that may be suitable for alkoxylation tooccur include those falling into one or more of the following ranges:from 10 psi to 1000 psi; from 15 psi to 500 psi; from 20 psi to 250 psi;from 25 psi to 100 psi. In addition, the alkoxylation pressure can beabout atmospheric pressure at sea level (e.g., 14.696 pounds per squareinch+/−10%).

In some instances, the alkoxylation conditions include addition of theoxirane compound in liquid form. In some instances, the alkoxylationconditions include addition of the oxirane compound in vapor form.

The alkoxylation conditions can include the use of a suitable solvent.Optimally, the system in which the alkoxylation conditions occur willnot include any component (including any solvent) that can bedeprotonated (or remains substantially protonated under the conditionsof pH, temperature, and so forth under which the alkoxylation conditionswill occur). Suitable solvents for alkoxylation include organic solventsselected from the group consisting of, tetrahydrofuran (THF),dimethylformamide (DMF), toluene, benzene, xylenes, mesitylene,tetrachloroethylene, anisole, dimethylacetamide, and mixtures of theforegoing. Less ideal solvents (but nonetheless still contemplated) foruse as part of the alkoxylation conditions are acetonitrile,phenylacetonitrile and ethyl acetate; in some instances, thealkoxylation conditions will not include as a solvent any ofacetonitrile, phenylacetonitrile and ethyl acetate.

In one or more embodiments of the invention, when the alkoxylationconditions are conducted in the liquid phase, the alkoxylationconditions are conducted such that both the alkoxylatable oligomer andthe desired alkoxylated polymeric material formed from alkoxylating thealkoxylatable oligomer not only have similar solubilities (and,preferably, substantially the same solubility) in the suitable solventused, but are also both substantially soluble in the suitable solvent.For example, in one or more embodiments, the alkoxylatable oligomer willbe substantially soluble in the solvent used in the alkoxylationconditions and the resulting alkoxylated polymeric material also will besubstantially soluble in the alkoxylation conditions.

In one or more embodiments, this substantially same solubility of thealkoxylated oligomer and the alkoxylated polymeric material in asuitable solvent stands in contrast to the solubility of a precursormolecule (used, for example, in the preparation of the previouslyisolated alkoxylated oligomer) in the suitable solvent, wherein theprecursor molecule can have a lower (and even substantially lower)solubility in the suitable solvent than the alkoxylated oligomer and/orthe alkoxylated polymeric material. By way of example only, thealkoxylated oligomer and the alkoxylated polymeric material will bothhave a pentaerthritol core and will both be substantially soluble intoluene, but pentaerthritol itself has limited solubility in toluene.

It is particularly preferred that the solvent employed in thealkoxylation conditions is toluene. The amount of toluene used for thereaction is greater than 25 wt % and less than 75 wt % of the reactionmixture, based on the weight of reaction mixture after complete additionof the oxirane compound. One of ordinary skill in the art can calculatethe starting amount of the solvent by taking into account the desiredmolecular weight of the polymer, the number of sites for whichalkoxylation will take place, the weight of the alkoxylatable oligomerused, and so forth.

It is preferred that the amount of the toluene is measured so that theamount is sufficient for the alkoxylation conditions providing thedesired alkoxylated polymeric material.

In addition, it is particularly preferred that the alkoxylationconditions have substantially no water present. Thus, it is preferredthat the alkoxylation conditions have a water content of less than 100ppm, more preferably 50 ppm, still more preferably 20 ppm, much morepreferably less than 14 ppm, and still even more preferably less than 8ppm.

The alkoxylation conditions take place in a suitable reaction vessel,typically a stainless steel reactor vessel.

In one or more embodiments, the alkoxylatable oligomer and/or precursormolecule lacks an isocyanate group attached to a carbon bearing an alphahydrogen is acceptable. In one or more embodiments, the previouslyprepared alkoxylatable oligomer and/or precursor molecule lacks anisocyanate group.

The Alkoxylatable Oligomer in the New Alkoxylation Method

The alkoxylatable oligomer used in the new alkoxylation method must haveat least one alkoxylatable functional group. The alkoxylatable oligomer,however, can have one, two, three, four, five, six, seven, eight or morealkoxylatable functional groups, with a preference for an alkoxylatableoligomer having from one to six alkoxylatable functional groups.

As stated previously, each alkoxylatable functional group within thealkoxylatable oligomer can be independently selected from the groupconsisting of hydroxyl, carboxylic acid, amine, thiol, aldehyde, ketone,and nitrile. In those instances where there is more than onealkoxylatable functional group within the alkoxylatable oligomer, it istypical that each alkoxylatable functional group is the same (e.g., eachalkoxylatable functional group within the alkoxylatable oligomer ishydroxyl), although instances of different alkoxylatable functionalgroups within the same alkoxylatable oligomer are contemplated as well.When the alkoxylatable functional group is hydroxyl, it is preferredthat the hydroxyl is a primary hydroxyl.

The alkoxylatable oligomer can take any of a number of possiblegeometries. For example, the alkoxylatable oligomer can be linear. Inone example of a linear alkoxylatable oligomer, one terminus of thelinear alkoxylatable oligomer is a relatively inert functional group(e.g., an end-capping group) and the other terminus is an alkoxylatablefunctional group (e.g., hydroxyl). An exemplary alkoxylatable oligomerof this structure is methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group. The structure of mPEG is given below.

CH₃O—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

(wherein, for the immediately preceding structure only, n is an integerfrom 13 to 100).

Another example of a linear geometry for which the alkoxylatableoligomer can take is a linear organic polymer bearing alkoxylatablefunctional groups (either the same or different) at each terminus. Anexemplary alkoxylatable oligomer of this structure is alpha-,omega-dihydroxylpoly(ethylene glycol), or

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

(wherein, for the immediately preceding structure only, n is an integerfrom 13 to 100),

which can be represented in brief form as HO-PEG-OH where it isunderstood that the -PEG- symbol represents the following structuralunit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

(wherein, for the immediately preceding structure only, n is an integerfrom 13 to 100).

Another geometry for which the alkoxylatable oligomer may have is a“multi-armed” or branched structure. With respect to such branchedstructures, one or more atoms in the alkoxylatable oligomer serves as a“branching point atom,” through which two, three, four or more (buttypically two, three or four) distinct sets of repeating monomers or“arms” are connected (either directly or through one or more atoms). Ata minimum, a “multi-arm” structure as used herein has three or moredistinct arms, but can have as many as four, five, six, seven, eight,nine, or more arms, with 4- to 8-arm multi-arm structures preferred(such as a 4-arm structure, a 5-arm structure, a 6-arm structure, and an8-arm structure).

Exemplary multi-arm structures for the alkoxylatable oligomer areprovided below:

wherein (for the immediately preceding structure only) the average valueof n is from 1 to 50, e.g., from 10 to 50, (or otherwise defined suchthat the molecular weight of the structure is from 300 Daltons to 9,000Daltons (e.g., from about 500 Daltons to 5,000 Daltons);

wherein (for the immediately preceding structure only) the average valueof n is from 2 to 50, e.g., from 10 to 50 (or otherwise defined suchthat the molecular weight of the structure is from 300 Daltons to 9,000Daltons (e.g., from about 500 Daltons to 5,000 Daltons);

wherein (for the immediately preceding structure only) the average valueof n is from 2 to 35, e.g., from 8 to about 40 (or otherwise definedsuch that the molecular weight of the structure is from 750 Daltons to9,500 Daltons (e.g., from 500 Daltons to 5,000 Daltons); and

wherein (for the immediately preceding structure only) the average valueof n is 2 to 35, e.g., from 5 to 35, (or otherwise defined such that themolecular weight of the structure is from 1,000 Daltons to 13,000Daltons (e.g., from 500 Daltons to 5,000 Daltons).

For each of the four immediately preceding structures, it is preferredthat the value of n, in each instance, is substantially the same. Thus,it is preferred that when all values of n are considered for a givenalkoxylatable oligomer, all values of n for that alkoxylatable oligomerare within three standard deviations, more preferably within twostandard deviations, and still more preferably within one standarddeviation.

In terms of the molecular weight of the alkoxylatable oligomer, thealkoxylatable oligomer will have a known and defined weight-averagemolecular weight. For use herein, a weight-average molecular weight canonly be known and defined for an alkoxylatable oligomer when thealkoxylatable oligomer is isolated from the synthetic milieu from whichit was generated. Exemplary weight-average molecular weights for thealkoxylatable oligomer will fall into one or more of the followingranges: greater than 300 Daltons; greater than 500 Daltons; from 300Daltons to 15,000 Daltons; from 500 Daltons to 5,000 Daltons; from 300Daltons to 10,000 Daltons; from 500 Daltons to 4,000 Daltons; from 300Daltons to 5,000 Daltons; from 500 Daltons to 3,000 Daltons; from 300Daltons to 2,000 Daltons; from 500 Daltons to 2,000 Daltons; from 300Daltons to 1,000 Daltons; from 500 Daltons to 1,000 Daltons; from 1,000Daltons to 10,000 Daltons; from 1,000 Daltons to 5,000 Daltons; from1,000 Daltons to 4,000 Daltons; from 1,000 Daltons to 3,000 Daltons;from 1,000 Daltons to 2,000 Daltons; from 1,500 Daltons to 15,000Daltons; from 1,500 Daltons to 5,000 Daltons; from 1,500 Daltons to10,000 Daltons; from 1,500 Daltons to 4,000 Daltons; from 1,500 Daltonsto 3,000 Daltons; from 1,500 Daltons to 2,000 Daltons; from 2,000Daltons to 5,000 Daltons; from 2,000 Daltons to 4,000 Daltons; and from2,000 Daltons to 3,000 Daltons.

For purposes of the present invention, the alkoxylatable oligomer ispreferably previously isolated. By previously isolated is meant thealkoxylatable oligomer exists outside and separate from the syntheticmilieu from which it was generated (most typically outside of thealkoxylating conditions used to prepare the alkoxylatable oligomer) andcan optionally be stored for a relatively long period of time oroptionally stored over a shorter time without substantially changing forsubsequent use. Thus, an alkoxylatable oligomer is previously isolatedif, for example, it is housed in an inert environment. In this regard, apreviously isolated alkoxylated oligomer can be housed in a containersubstantially lacking (e.g., less than 0.1 wt %) an oxirane compound.Also, a previously isolated alkoxylatable oligomer does not change itsmolecular weight more than 10% over the course of 15 days. Thus, in oneor more embodiments of the invention, the concept of “previouslyisolated” stands in contrast to (for example) a situation where anongoing and uninterrupted alkoxylation reaction is allowed to proceedfrom precursor molecule, into a structure that corresponds analkoxylatable oligomer, to a structure that corresponds to analkoxylated polymeric material; the concept of “previously isolated”requires that the alkoxylatable oligomer exists apart from theconditions from which it formed. Pursuant to the present invention,however, the previously isolated alkoxylatable oligomer will besubjected to an alkoxylation step once it is added to, as a separatestep, alkoxylation conditions.

Sources of the Alkoxylatable Oligomer in the New Alkoxylation Method

The alkoxylatable oligomer can be obtained via synthetic means. In thisregard, the alkoxylatable oligomer is prepared by (a) alkoxylating aprecursor molecule having a molecular weight of less than 300 Daltons(e.g., less than 500 Daltons) to form a reaction mixture comprising analkoxylatable oligomer or prepolymer, and (b) isolating thealkoxylatable oligomer from the reaction mixture. The step ofalkoxylating the precursor molecule largely follows the conditions andrequirements of the alkoxylating step previously discussed. The step ofisolating the alkoxylatable oligomer can be carried out using any artknown step, but can include allowing all oxirane compound to be consumedin the reaction, actively performing a quenching step, separating thefinal reaction mixture through art-known approaches (including, forexample, distilling off all volatile materials, removing solid reactionby-product by filtration or washing and applying chromatographic means).

In addition, the alkoxylatable oligomer can be obtained from commercialsources. Exemplary commercial sources include NOF Corporation (TokyoJapan) which provides alkoxylatable oligomers under the names SUNBRIGHTDKH® poly(ethylene glycol), SUNBRIGHT® GL glycerine, tri-poly(ethyleneglycol) ether, SUNBRIGHT PTE® pentaerythritol, tetra-poly(ethyleneglycol) ether. SUNBRIGHT® DG di-glycerine, tetra-poly(ethylene glycol)ether, and SUNBRIGHT HGEO® hexa-glycerine, octa-poly(ethylene glycol)ether. Preferred alkoxylatable oligomers include those having thestructures of SUNBRIGHT PTE®-2000 pentaerythritol, tetra-poly(ethyleneglycol) ether (which has a weight-average molecular weight of about2,000 Daltons) and SUNBRIGHT® DG-2000 di-glycerine, tetra-poly(ethyleneglycol) ether (which has a weight-average molecular weight of about2,000 Daltons).

Precursor molecules can be any small molecule (e.g., a molecular weightless than the weight-average molecular weight of the alkoxylatableoligomer) having one or more alkoxylatable functional groups.

Exemplary precursor molecules include polyols, which are small molecules(typically of a molecular weight of less than 300 Daltons, e.g., lessthan 500 Daltons) having a plurality of available hydroxyl groups.Depending on the desired number of polymer arms in the alkoxylatableoligomer or prepolymer, the polyol serving as the precursor moleculewill typically comprise 3 to about 25 hydroxyl groups, preferably about3 to about 22 hydroxyl groups, most preferably about 4 to about 12hydroxyl groups. Preferred polyols include glycerol oligomers orpolymers such as hexaglycerol, pentaerythritol and oligomers or polymersthereof (e.g., dipentaerythritol, tripentaerythritol,tetrapentaerythritol, and ethoxylated forms of pentaerythritol), andsugar-derived alcohols such as sorbitol, arabanitol, and mannitol. Also,many commercially available polyols, such as various isomers of inositol(i.e. 1,2,3,4,5,6-hexahydroxycyclohexane),2,2-bis(hydroxymethyl)-1-butanol,2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol,{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}acetic acid (Tricine),2-[(3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}propyl)amino]-2-(hydroxymethyl)-1,3-propanediol,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid(TES), 4-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-butanesulfonicacid, and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediolhydrochloride can serve as an acceptable precursor molecule. In thosecases in which the precursor molecule has an ionizable group or groupsthat will interfere with the alkoxylation step, those ionizable groupsmust be protected or modified prior to carrying out the alkoxylationstep.

Exemplary preferred precursor molecules include those precursormolecules selected from the group consisting of glycerol, diglycerol,triglycerol, hexaglycerol, mannitol, sorbitol, pentaerythritol,dipentaerthitol, and tripentaerythritol.

In one or more embodiments of the invention, it is preferred thatneither the previously isolated alkoxylatable oligomer nor thealkoxylated polymeric product has an alkoxylatable functional group(e.g., hydroxyl group) of the precursor molecule.

The Alkoxylated Polymeric Materials Generated by the New AlkoxylationMethod

The alkoxylated polymeric material prepared under the methods describedherein will have a basic architecture corresponding to the structure ofthe alkoxylatable oligomer (i.e., a linear alkoxylatable oligomerresults in a linear alkoxylated polymeric material, a four-armedalkoxylatable oligomer results in a four-armed alkoxylated polymermaterial, so forth). As a consequence, the alkoxylated polymericmaterial will take any of a number of possible geometries, includinglinear, branched and multi-armed.

With respect to branched structures, a branched alkoxylated polymericmaterial will have three or more distinct arms, but can have as many asfour, five, six, seven, eight, nine, or more arms, with 4- to 8-armbranched structures preferred (such as a 4-arm branched structure, 5-armbranched structure, 6-arm branched structure, and 8-arm branchedstructure).

Exemplary branched structures for the alkoxylated polymeric material areprovided below:

wherein (for the immediately preceding structure only) the average valueof n satisfies one or more of the following ranges: from 10 to 1,000;from 10 to 500; from 10 to 250; from 50 to 1000; from 50 to 250; andfrom 50 to 120 (or otherwise defined such that the molecular weight ofthe structure is from 2,000 Daltons to 180,000 Daltons, e.g., from 2,000Daltons to 120,000 Daltons);

wherein (for the immediately preceding structure only) the average valueof n satisfies one or more of the following ranges: from 10 to 1,000;from 10 to 500; from 10 to 250; from 50 to 1,000; from 50 to 250; andfrom 50 to 120 (or otherwise defined such that the molecular weight ofthe structure is from 2,000 Daltons to 180,000 Daltons, e.g., from 2,000Daltons to 120,000 Daltons);

wherein (for the immediately preceding structure only) the average valueof n is satisfies one or more of the following ranges: from 10 to 750;from 40 to 750; from 50 to 250; and from 50 to 120 (or otherwise definedsuch that the molecular weight of the structure is from 3.000 Daltons to200,000 Daltons, e.g., from 12,000 Daltons to 200,000 Daltons); and

wherein (for the immediately preceding structure only) the average valueof n is satisfies one or more of the following ranges: from 10 to 600and from 35 to 600 (or otherwise defined such that the molecular weightof the structure is from 4,000 Daltons to 215,000 Daltons, e.g., from12,000 Daltons to 215,000 Daltons).

For each of the four immediately provided structures, it is preferredthat the value of n, in each instance, is substantially the same. Thus,it is preferred that when all values of n are considered for a givenalkoxylated polymeric material, all values of n for that alkoxylatedpolymeric material alkoxylatable oligomer or prepolymer are within threestandard deviations, more preferably within two standard deviations, andstill more preferably within one standard deviation.

In terms of the molecular weight of the alkoxylated polymeric material,the alkoxylated polymeric material will have a known and definednumber-average molecular weight. For use herein, a number-averagemolecular weight can only be known and defined for material that isisolated from the synthetic milieu from which it was generated.

The total molecular weight of the alkoxylated polymeric product can be amolecular weight suited for the intended purpose. An acceptablemolecular weight for any given purpose can be determined through trialand error via routine experimentation. Exemplary molecular weights forthe alkoxylated polymeric product, will have a number-average molecularweight falling within one or more of the following ranges: from 2,000Daltons to 215,000 Daltons; from 5,000 Daltons to 215,000 Daltons; from5,000 Daltons to 150,000 Daltons; from 5,000 Daltons to 100,000 Daltons;from 5,000 Daltons to 80,000 Daltons; from 6,000 Daltons to 80,000Daltons; from 7,500 Daltons to 80,000 Daltons; from 9,000 Daltons to80,000 Daltons; from 10,000 Daltons to 80,000 Daltons; from 12,000Daltons to 80,000 Daltons; from 15,000 Daltons to 80.000 Daltons; from20,000 Daltons to 80,000 Daltons; from 25,000 Daltons to 80,000 Daltons;from 30,000 Daltons to 80,000 Daltons; from 40,000 Daltons to 80,000Daltons; from 6,000 Daltons to 60,000 Daltons; from 7,500 Daltons to60,000 Daltons; from 9,000 Daltons to 60,000 Daltons; from 10,000Daltons to 60,000 Daltons; from 12,000 Daltons to 60,000 Daltons; from15,000 Daltons to 60,000 Daltons; from 20,000 Daltons to 60,000 Daltons;from 25,000 Daltons to 60,000 Daltons; from 30,000 Daltons to 60,000;from 6,000 Daltons to 40,000 Daltons; from 9,000 Daltons to 40,000Daltons; from 10,000 Daltons to 40,000 Daltons; from 15,000 Daltons to40,000 Daltons; from 19,000 Daltons to 40,000 Daltons; from 15,000Daltons to 25,000 Daltons; and from 18,000 Daltons to 22,000 Daltons.

For any given alkoxylated polymeric material, an optional step can becarried out so as to further transform the alkoxylated polymericmaterial so that it bears a specific reactive group to form a polymericreagent. Thus, using techniques well known in the art, the alkoxylatedpolymeric material can be functionalized to include a reactive group(e.g., carboxylic acid, active ester, amine, thiol, maleimide, aldehyde,ketone, and so forth).

In carrying out an optional step to further transform the alkoxylatedpolymeric product so that it bears a specific reactive group, such anoptional step is carried out in a suitable solvent. One of ordinaryskill in the art can determine whether any specific solvent isappropriate for any given reaction step. Often, however, the solvent ispreferably a nonpolar solvent or a polar solvent. Nonlimiting examplesof nonpolar solvents include benzene, xylenes and toluene. Exemplarypolar solvents include, but are not limited to, dioxane, tetrahydrofuran(THF), t-butyl alcohol, DMSO (dimethyl sulfoxide). HMPA(hexamethylphosphoramide). DMF (dimethylformamide), DMA(dimethylacetamide), and NMP (N-methylpyrrolidinone).

Further Compositions of the Alkoxylated Polymeric Material

Another aspect of the invention provided herein are compositionscomprising the alkoxylated polymeric material, which include not onlyany compositions comprising the alkoxylated polymeric material, but alsocompositions in which the alkoxylated polymeric material is furthertransformed into, for example, a polymer reagent, as well ascompositions of conjugates formed from coupling such polymer reagentswith an active agent. Among other things, a benefit of the methoddescribed herein is the ability to achieve high purity alkoxylatedpolymeric material-containing compositions. The compositions can becharacterized as having: substantially low content of both highmolecular weight impurities (e.g. polymer-containing species having amolecular weight greater than the molecular weight of the desiredalkoxylated polymeric material) and low content of low molecular weightdiol impurities (i.e., HO-PEG-OH), either impurity type (and preferablyboth impurity types) totaling less than 8 wt %, and more preferably lessthan 2 wt %. In addition or alternatively, the compositions can also becharacterized as having a purity of alkoxylated polymeric material (aswell as compositions comprising polymer reagents formed from thealkoxylated polymeric material, and compositions of conjugates formedfrom conjugating such polymer reagents and an active agent) of greaterthan 92 wt %, more preferably greater than 97 wt %. Gel pearmeationchromatography (GPC) and gel filtration chromatography (GFC) can be usedto characterize the alkoxylated polymeric material. Thosechromatographic methods allow separation of the composition to itscomponents according to molecular weight. The exemplary GFC traces ofproducts described in Example 7 and Example 8 are provided as FIG. 7 andFIG. 8, respectively.

Exemplary Uses of the Alkoxylated Polymeric Materials and CompositionsFormed Therefrom

The alkoxylated polymeric material provided herein as well as thosealkoxylated polymeric products that have been further modified to bear aspecific reactive group (hereinafter referred to as a “polymer reagent”)are useful for conjugation to, for example, active agents. Preferredgroups of the biologically active agents suited for reaction with thepolymeric reagents described herein are electrophilic and nucleophilicgroups. Exemplary groups include primary amines, carboxylic acids,alcohols, thiols, hydrazines and hydrazides. Such groups suited to reactwith the polymeric reagents described herein are known to those ofordinary skill in the art. Thus, the invention provides a method formaking a conjugate comprising the step of contacting, under conjugationconditions, an active agent with a polymeric reagent described herein.

Suitable conjugation conditions are those conditions of time,temperature, pH, reagent concentration, reagent functional group(s),available functional groups on the active agent, solvent, and the likesufficient to effect conjugation between a polymeric reagent and anactive agent. As is known in the art, the specific conditions dependupon, among other things, the active agent, the type of conjugationdesired, the presence of other materials in the reaction mixture, and soforth. Sufficient conditions for effecting conjugation in any particularcase can be determined by one of ordinary skill in the art upon areading of the disclosure herein, reference to the relevant literature,and/or through routine experimentation.

For example, when the polymeric reagent contains an N-hydroxysuccinimideactive ester (e.g., succinimidyl succinate, succinimidyl propionate, andsuccinimidyl butanoate), and the active agent contains an amine group,conjugation can be effected at a pH of from about 7.5 to about 9.5 atroom temperature. In addition, when the polymer reagent contains avinylsulfone reactive group or a maleimide group and thepharmacologically active agent contains a sulfhydryl group, conjugationcan be effected at a pH1 of from about 7 to about 8.5 at roomtemperature. Moreover, when the reactive group associated with thepolymer reagent is an aldehyde or ketone and the pharmacologicallyactive agent contains a primary amine, conjugation can be effected byreductive amination wherein the primary amine of the pharmacologicallyactive agent reacts with the aldehyde or ketone of the polymer. Takingplace at pH's of from about 6 to about 9.5, reductive aminationinitially results in a conjugate wherein the pharmacologically activeagent and polymer are linked via an imine bond. Subsequent treatment ofthe imine bond-containing conjugate with a suitable reducing agent suchas NaCNBH₃ reduces the imine to a secondary amine. For additionalinformation concerning these and other conjugation reactions, referenceis made to Hermanson “Bioconjugate Techniques,” Academic Press, 1996.

Exemplary conjugation conditions include carrying out the conjugationreaction at a pH of from about 4 to about 10, and at, for example, a pH1of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or10.0. The reaction is allowed to proceed from about 5 minutes to about72 hours, preferably from about 30 minutes to about 48 hours, and morepreferably from about 4 hours to about 24 hours. The temperature underwhich conjugation can take place is typically, although not necessarily,in the range of from about 0° C. to about 40° C., and is often at roomtemperature or less. The conjugation reactions are often carried outusing a phosphate buffer solution, sodium acetate, or similar system.

With respect to reagent concentration, an excess of the polymer reagentis typically combined with the active agent. In some cases, however, itis preferred to have stoichiometric amounts of reactive groups on thepolymer reagent to the reactive groups of the active agent. Thus, forexample, one mole of a polymer reagent bearing four reactive groups iscombined with four moles of active agent. Exemplary ratios of reactivegroups of polymer reagent to active agent include molar ratios of about1:1 (reactive group of polymer reagent:active agent), 1:0.1, 1:0.5,1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10. The conjugation reactionis allowed to proceed until substantially no further conjugation occurs,which can generally be determined by monitoring the progress of thereaction over time.

Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by chromatographic methods, SDS-PAGE or MALDI-TOF massspectrometry, NMR, IR, or any other suitable analytical method. Once aplateau is reached with respect to the amount of conjugate formed or theamount of unconjugated polymer reagent remaining, the reaction isassumed to be complete. Typically, the conjugation reaction takesanywhere from minutes to several hours (e.g., from 5 minutes to 24 hoursor more). The resulting product mixture is preferably, but notnecessarily purified, to separate out excess active agent, strong base,condensing agents and reaction by-products and solvents. The resultingconjugates can then be further characterized using analytical methodssuch as chromatographic methods, spectroscopic methods, MALDI, capillaryelectrophoresis, and/or gel electrophoresis. The polymer-active agentconjugates can be purified to obtain/isolate different conjugatedspecies.

With respect to an active agent, the alkoxylated polymeric material anda polymer reagent prepared from the alkoxylated polymeric material canbe combined under suitable conjugation conditions to result in aconjugate. In this regard, exemplary active agents can be an activeagent selected from the group consisting of a small molecule drug, anoligopeptide, a peptide, and a protein. The active agent for use hereincan include but are not limited to the following: adriamycin,γ-aminobutyric acid (GABA), amiodarone, amitryptyline, azithromycin,benzphetamine, bromopheniramine, cabinoxamine, calcitonin chlorambucil,chloroprocaine, chloroquine, chlorpheniramine, chlorpromazine,cinnarizine, clarthromycin, clomiphene, cyclobenzaprine, cyclopentolate,cyclophosphamide, dacarbazine, daunomycin, demeclocycline, dibucaine,dicyclomine, diethylproprion, diltiazem, dimenhydrinate,diphenhydramine, disopyramide, doxepin, doxycycline, doxylamine,dypyridame, EDTA, erythromycin, flurazepam, gentian violet,hydroxychloroquine, imipramine, insulin, irinotecan, levomethadyl,lidocaine, loxarine, mechlorethamine, melphalan, methadone,methotimeperazine, methotrexate, metoclopramide, minocycline, naftifine,nicardipine, nizatidine, orphenadrine, oxybutin, oxytetracycline,phenoxybenzamine, phentolamine, procainamide, procaine, promazine,promethazine, proparacaine, propoxycaine, propoxyphene, ranitidine,tamoxifen, terbinafine, tetracaine, tetracycline, tranadol,triflupromazine, trimeprazine, trimethylbenzamide, trimipramine,trlpelennamine, troleandomycin, tyramine, uracil mustard, verapamil, andvasopressin.

Further exemplary active agents include those selected from the groupconsisting of acravistine, amoxapine, astemizole, atropine,azithromycin, benzapril, benztropine, beperiden, bupracaine,buprenorphine, buspirone, butorphanol, caffeine, camptothecin andmolecules belonging to the camptothecin family, cefiriaxone,chlorpromazine, ciprofloxacin, cladarabine, clemastine, clindamycin,clofazamine, clozapine, cocaine, codeine, cyproheptadine, desipramine,dihydroergotamine, diphenidol, diphenoxylate, dipyridamole, docetaxel,doxapram, ergotamine, famciclovir, fentanyl, flavoxate, fludarabine,fluphenazine, fluvastin, ganciclovir, granisteron, guanethidine,haloperidol, homatropine, hydrocodone, hydromorphone, hydroxyzine,hyoscyamine, imipramine, itraconazole, keterolac, ketoconazole,levocarbustine, levorphone, lincomycin, lomefloxacin, loperamide,losartan, loxapine, mazindol, meclizine, meperidine, mepivacaine,mesoridazine, methdilazine, methenamine, methimazole,methotrimeperazine, methysergide, metronidazole, minoxidil, mitomycin c,molindone, morphine, nafzodone, nalbuphine, naldixic acid, nalmefene,naloxone, naltrexone, naphazoline, nedocromil, nicotine, norfloxacin,ofloxacin, ondansteron, oxycodone, oxymorphone, paclitaxel, pentazocine,pentoxyfylline, perphenazine, physostigmine, pilocarpine, pimozide,pramoxine, prazosin, prochlorperazine, promazine, promethazine,quinidine, quinine, rauwolfia alkaloids, riboflavin, rifabutin,risperidone, rocuronium, scopalamine, sufentanil, tacrine, terazosin,terconazole, terfenadine, thiordazine, thiothixene, ticlodipine,timolol, tolazamide, tolmetin, trazodone, triethylperazine,trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine,tubocurarine, vecuronium, vidarabine, vinblastine, vincristine andvinorelbine.

Still further exemplary active agents include those selected from thegroup consisting of acetazolamide, acravistine, acyclovir, adenosinephosphate, allopurinal, alprazolam, amoxapine, aminone, apraclonidine,azatadine, aztreonam, bisacodyl, bleomycin, bromopheniramine, buspirone,butoconazole, camptothecin and molecules within the camptothecin family,carbinoxamine, cefamandole, cefazole, cefixime, cefmetazole, cefonicid,cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone,cephapirin, chloroquine, chlorpheniramine, cimetidine, cladarabine,clotrimazole, cloxacillin, didanosine, dipyridamole, doxazosin,doxylamine, econazole, enoxacin, estazolam, ethionamide, famciclovir,famotidine, fluconazole, fludarabine, folic acid, ganciclovir,hydroxychloroquine, iodoquinol, isoniazid, itraconazole, ketoconazole,lamotrigine, lansoprazole, lorcetadine, losartan, mebendazole,mercaptopurine, methotrexate, metronidazole, miconazole, midazolam,minoxidil, nafzodone, naldixic acid, niacin, nicotine, nizatidine,omeperazole, oxaprozin, oxiconazole, papaverine, pentostatin,phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine,pyrazinamide, pyrimethamine, pyroxidine, quinidine, quinine, ribaverin,rifampin, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfasalazine,sulfasoxazole, terazosin, thiabendazole, thiamine, thioguanine, timolol,trazodone, triampterene, triazolam, trimethadione, trimethoprim,trimetrexate, triplenamine, tropicamide, and vidarabine.

Still further exemplary active agents include those belonging to thecamptothecin family of molecules. For example, the active agent canpossess the general structure:

wherein R₁, R₂, R₃, R₄ and R₅ are each independently selected from thegroup consisting of: hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl);substituted alkyl; alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy;alkenyl; alkynyl; cycloalkyl; hydroxyl; cyano: nitro; azido; amido;hydrazine; amino; substituted amino (e.g., monoalkylamino anddialkylamino); hydroxcarbonyl; alkoxycarbonyl: alkylcarbonyloxy;alkylcarbonylamino; carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy;—C(R₇)═N—(O)_(i)—R₈ wherein R₇ is H, alkyl, alkenyl, cycloalkyl, oraryl, i is 0 or 1, and R₈ is H, alkyl, alkenyl, cycloalkyl, orheterocycle; and R₉C(O)O— wherein R₉ is halogen, amino, substitutedamino, heterocycle, substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where mis an integer of 1-10 and R₁₀ is alkyl, phenyl, substituted phenyl,cycloalkyl, substituted cycloalkyl, heterocycle, or substitutedheterocycle; or R₂ together with R₃ or R₃ together with R₄ formsubstituted or unsubstituted methylenedioxy, ethylenedioxy, orethyleneoxy; R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl,haloalkyl, or hydroxyalkyl. Although not shown, analogs having ahydroxyl group corresponding to a position other than the 20-position(e.g., 10-, or 11-position, and so forth) in the immediately precedingstructure are encompassed within possible active agents.

An exemplary active agent is irinotecan.

Another exemplary active agent is 7-ethyl-10-hydroxy-camptothecin(SN-38), the structure of which is shown below.

Yet other exemplary class of active agents include those belonging tothe taxane family of molecules. An exemplary active agent from thisclass of molecules is docetaxel, where the H at the 2′ hydroxyl group isinvolved in forming the preferred multi-armed polymer conjugate:

The polymer reagents described herein can be attached, either covalentlyor noncovalently, to a number of entities including films, chemicalseparation and purification surfaces, solid supports, metal surfacessuch as gold, titanium, tantalum, niobium, aluminum, steel, and theiroxides, silicon oxide, macromolecules (e.g., proteins, polypeptides, andso forth), and small molecules. Additionally, the polymer reagents canalso be used in biochemical sensors, bioelectronic switches, and gates.The polymer reagents can also be employed as carriers for peptidesynthesis, for the preparation of polymer-coated surfaces and polymergrafts, to prepare polymer-ligand conjugates for affinity partitioning,to prepare cross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

Optionally, the conjugate can be provided as a pharmaceuticalcomposition for veterinary and for human medical use. Such apharmaceutical compositions is prepared by combining the conjugate withone or more pharmaceutically acceptable excipients, and optionally anyother therapeutic ingredients.

Exemplary pharmaceutically acceptable excipients, without limitation,those selected from the group consisting of carbohydrates, inorganicsalts, antimicrobial agents, antioxidants, surfactants, buffers, acids,bases, and combinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The composition can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for one or more embodiments of the present inventioninclude benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in one or more embodiments of the present inventioninclude, for example, ascorbyl palmitate, butylated hydroxyanisole,butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite, and combinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymeric reagent) in the composition will varydepending on a number of actors, but will optimally be a therapeuticallyeffective dose when the composition is stored in a unit dose container(e.g., a vial). In addition, the pharmaceutical preparation can behoused in a syringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about 5%to about 98% by weight, more preferably from about 15 to about 95% byweight of the excipient, with concentrations less than 30% by weightmost preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutically acceptable compositions encompass all types offormulations and in particular those that are suited for injection,e.g., powders or lyophilates that can be reconstituted as well asliquids. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof. With respect to liquid pharmaceutical compositions, solutionsand suspensions are envisioned.

The compositions of one or more embodiments of the present invention aretypically, although not necessarily, administered via injection and aretherefore generally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering to a patient, generally via injection, a therapeuticallyeffective amount of the conjugate (preferably provided as part of apharmaceutical composition). As previously described, the conjugates canbe administered injected parenterally by intravenous injection. Suitableformulation types for parenteral administration includeready-for-injection solutions, dry powders for combination with asolvent prior to use, suspensions ready for injection, dry insolublecompositions for combination with a vehicle prior to use, and emulsionsand liquid concentrates for dilution prior to administration, amongothers.

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate can effectively treat. Advantageously, the conjugate can beadministered to the patient prior to, simultaneously with, or afteradministration of another active agent.

The actual dose to be administered will vary depending upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day. A given dose can beperiodically administered up until, for example, symptoms lessen and/orare eliminated entirely.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering certain conjugates described herein isthat individual water-soluble polymer portions can be cleaved when ahydrolytically degradable linkage is included between the residue of theactive agent moiety and water-soluble polymer. Such a result isadvantageous when clearance from the body is potentially a problembecause of the polymer size. Optimally, cleavage of each water-solublepolymer portion is facilitated through the use of physiologicallycleavable and/or enzymatically degradable linkages such as amide,carbonate or ester-containing linkages. In this way, clearance of theconjugate (via cleavage of individual water-soluble polymer portions)can be modulated by selecting the polymer molecular size and the typefunctional group that would provide the desired clearance properties.One of ordinary skill in the art can determine the proper molecular sizeof the polymer as well as the cleavable functional group. For example,one of ordinary skill in the art, using routine experimentation, candetermine a proper molecular size and cleavable functional group byfirst preparing a variety of polymer derivatives with different polymerweights and cleavable functional groups, and then obtaining theclearance profile (e.g., through periodic blood or urine sampling) byadministering the polymer derivative to a patient and taking periodicblood and/or urine sampling. Once a series of clearance profiles havebeen obtained for each tested conjugate, a suitable conjugate can beidentified.

Hydrohalide Salts Considerations Concerning the Active Agent, “D”

The hydrohalide salt compositions described herein comprise awater-soluble polymer-active agent conjugate, preferably a multi-armedpolymer bioactive conjugate. Exemplary water soluble polymers aredescribed above. Turning now to the active agent, the active agent is asmall molecule drug, an oligopeptide, a peptide, or a protein. Theactive agent, when conjugated to the water-soluble polymer, contains atleast one basic nitrogen atom such as an amine group (i.e., an amine orother basic nitrogen containing group that is not conjugated to thewater-soluble polymer). In the hydrohalide salt, the basic nitrogenatoms are in protonated form as the hydrohalide salt, that is to say,where at least 90 mole percent, or at least 91 mole percent, or at least92 mole percent, or at least 93 mole percent, or at least 94 molepercent, or at least 95 mole percent, more preferably greater than 95mole percent of the drug's basic nitrogen atoms within the conjugate areprotonated in HX form.

Active agents containing at least one amine group or basic nitrogen atomsuitable for providing a mixed acid salt as described herein include butare not limited to the following: adriamycin, γ-aminobutyric acid(GABA), amiodarone, amitryptyline, azithromycin, benzphetamine,bromopheniramine, cabinoxamine, calcitonin chlorambucil, chloroprocaine,chloroquine, chlorpheniramine, chlorpromazine, cinnarizine,clarthromycin, clomiphene, cyclobenzaprine, cyclopentolate,cyclophosphamide, dacarbazine, daunomycin, demeclocycline, dibucaine,dicyclomine, diethylproprion, diltiazem, dimenhydrinate,diphenhydramine, disopyramide, doxepin, doxycycline, doxylamine,dypyridame, EDTA, erythromycin, flurazepam, gentian violet,hydroxychloroquine, imipramine, insulin, irinotecan, levomethadyl,lidocaine, loxarine, mechlorethamine, melphalan, methadone,methotimeperazine, methotrexate, metoclopramide, minocycline, naftifine,nicardipine, nizatidine, orphenadrine, oxybutin, oxytetracycline,phenoxybenzamine, phentolamine, procainamide, procaine, promazine,promethazine, proparacaine, propoxycaine, propoxyphene, ranitidine,tamoxifen, terbinafine, tetracaine, tetracycline, tranadol,triflupromazine, trimeprazine, trimethylbenzamide, trimipramine,trlpelennamine, troleandomycin, tyramine, uracil mustard, verapamil, andvasopressin.

Additional active agents include those comprising one or morenitrogen-containing heterocycles such as acravistine, amoxapine,astemizole, atropine, azithromycin, benzapril, benztropine, beperiden,bupracaine, buprenorphine, buspirone, butorphanol, caffeine,camptothecin and molecules belonging to the camptothecin family,ceftriaxone, chlorpromazine, ciprofloxacin, cladarabine, clemastine,clindamycin, clofazamine, clozapine, cocaine, codeine, cyproheptadine,desipramine, dihydroergotamine, diphenidol, diphenoxylate, dipyridamole,doxapram, ergotamine, famciclovir, fentanyl, flavoxate, fludarabine,fluphenazine, fluvastin, ganciclovir, granisteron, guanethidine,haloperidol, homatropine, hydrocodone, hydromorphone, hydroxyzine,hyoscyamine, imipramine, itraconazole, keterolac, ketoconazole,levocarbustine, levorphone, lincomycin, lomefloxacin, loperamide,losartan, loxapine, mazindol, meclizine, meperidine, mepivacaine,mesoridazine, methdilazine, methenamine, methimazole,methotrimeperazine, methysergide, metronidazole, minoxidil, mitomycin c,molindone, morphine, nafzodone, nalbuphine, naldixic acid, nalmefene,naloxone, naltrexone, naphazoline, nedocromil, nicotine, norfloxacin,ofloxacin, ondansteron, oxycodone, oxymorphone, pentazocine,pentoxyfylline, perphenazine, physostigmine, pilocarpine, pimozide,pramoxine, prazosin, prochlorperazine, promazine, promethazine,quinidine, quinine, rauwolfia alkaloids, riboflavin, rifabutin,risperidone, rocuronium, scopalamine, sufentanil, tacrine, terazosin,terconazole, terfenadine, thiordazine, thiothixene, ticlodipine,timolol, tolazamide, tolmetin, trazodone, triethylperazine,trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine,tubocurarine, vecuronium, vidarabine, vinblastine, vincristine andvinorelbine.

Additional active agents include those comprising an aromatic ringnitrogen such as acetazolamide, acravistine, acyclovir, adenosinephosphate, allopurinal, alprazolam, amoxapine, aminone, apraclonidine,azatadine, aztreonam, bisacodyl, bleomycin, bromopheniramine, buspirone,butoconazole, camptothecin and molecules within the camptothecin family,carbinoxamine, cefamandole, cefazole, cefixime, cefmetazole, cefonicid,cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone,cephapirin, chloroquine, chlorpheniramine, cimetidine, cladarabine,clotrimazole, cloxacillin, didanosine, dipyridamole, doxazosin,doxylamine, econazole, enoxacin, estazolam, ethionamide, famciclovir,famotidine, fluconazole, fludarabine, folic acid, ganciclovir,hydroxychloroquine, iodoquinol, isoniazid, itraconazole, ketoconazole,lamotrigine, lansoprazole, lorcetadine, losartan, mebendazole,mercaptopurine, methotrexate, metronidazole, miconazole, midazolam,minoxidil, nafzodone, naldixic acid, niacin, nicotine, nizatidine,omeperazole, oxaprozin, oxiconazole, papaverine, pentostatin,phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine,pyrazinamide, pyrimethamine, pyroxidine, quinidine, quinine, ribaverin,rifampin, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfasalazine,sulfasoxazole, terazosin, thiabendazole, thiamine, thioguanine, timolol,trazodone, triampterene, triazolam, trimethadione, trimethoprim,trimetrexate, triplenamine, tropicamide, and vidarabine.

A preferred active agent is one belonging to the camptothecin family ofmolecules. For example, the active agent may possess the generalstructure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; R₆ is H orOR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl.

In reference to the foregoing structure, although not shown, analogshaving a hydroxyl group at other than the 20-position (e.g., 10-, or11-position, etc.) are similarly preferred.

In one particular embodiment, the active agent is irinotecan (structureshown immediately below),

In another embodiment, the active agent is irinotecan having a glycinelinker at the 20-hydroxyl position (structure shown immediately below),

In yet another particular embodiment, the active agent is7-ethyl-10-hydroxy-camptothecin (SN-38), a metabolite of irinotecan,whose structure is shown below.

In the foregoing embodiment, covalent attachment of the active agent,SN-38, to the multi-armed polymer core similarly occurs at the20-hydroxyl position, optionally via an intervening linker such asglycine, as shown below.

Hydrohalide Salts Considerations Concerning the Conjugates

Illustrative conjugates of a water-soluble polymer and an active agentmay possess any of a number of structural features as described above.That is to say, the conjugate may possess a linear structure, i.e.,having one or two active agent molecules covalently attached thereto,typically at the polymer terminus or termini, respectively.Alternatively, the conjugate may possess a forked, branched ormulti-armed structure. Preferably, the conjugate is a multi-armedpolymer conjugate.

One illustrative multi-armed polymer conjugate structure corresponds tothe following:

The foregoing structure is referred to herein in shorthand fashion as“4-arm-PEG-Gly-Irino” (4-arm-pentaerythritolyl-PEG-carboxymethylglycineirinotecan); a more complete name corresponds to“pentaerythritolyl-4-arm-(PEG-1-methylene-2-oxo-vinylamino acetatelinked-irinotecan).” Basic amino and/or nitrogen groups in the activeagent portion of the conjugate are shown above in only neutral form,with the understanding that the conjugate possesses the features of ahydrohalide salt (HX) as described in detail herein. As can be seen fromthe structure above, the carboxymethyl modified 4-arm pentaerythritolylPEG reagent possesses a glycine linker intervening between the polymerportion and the active agent, irinotecan.

Typically, although not necessarily, the number of polymer arms willcorrespond to the number of active agent molecules covalently attachedto the water-soluble polymer core. That is to say, in the case of apolymer reagent having a certain number of polymer arms (e.g.,corresponding to the variable “q”), each having a reactive functionalgroup (e.g., carboxy, activated ester such as succinimidyl ester,benzotriazolyl carbonate, and so forth) at its terminus, the optimizednumber of active agents (such as irinotecan) that can be covalentlyattached thereto in the corresponding conjugate is most desirably “q.”That is to say, the optimized conjugate is considered to have a drugloading value of 1.00(q) (or 100%). In a preferred embodiment, themulti-armed polymer conjugate is characterized by a degree of drugloading of 0.90(q) (or 90%) or greater. Preferred drug loadings satisfyone or more of the following: 0.92(q) or greater; 0.93(q) or greater;0.94(q) or greater; 0.95(q) or greater; 0.96(q) or greater; 0.97(q) orgreater: 0.98(q) or greater; and 0.99(q) or greater. Most preferably,the drug loading for a multi-armed polymer conjugate is one hundredpercent. A composition comprising a multi-arm water soluble polymerconjugate hydrohalide salt may comprise a mixture of molecularconjugates having one active agent attached to the polymer core, havingtwo active agent molecules attached to the polymer core, having threeactive agents attached to the polymer core, and so on, up to andincluding a conjugate having “q” active agents attached to the polymercore. The resulting composition will possess an overall drug loadingvalue, averaged over the conjugate species contained in the composition.Ideally, the composition will comprise a majority, e.g., greater than50%, but more preferably greater than 60%, still more preferably greaterthan 70%, still yet more preferably greater than 80%, and mostpreferably greater than 90%) of drug fully loaded polymer conjugates(i.e., having “q” active agent molecules for “q” arms, a single activeagent molecule for each arm).

As an illustration, in an instance in which the multi-armed polymerconjugate contains four polymer arms, the idealized value of the numberof covalently attached drug molecules per multi-armed polymer is four,and—with respect to describing the average in the context of acomposition of such conjugates—there will be a value (i.e., percentage)of drug molecules loaded onto multi-armed polymer ranging from about 90%to about 100% of the idealized value. That is to say, the average numberof drug molecules covalently attached to a given four-armed polymer (aspart of a four-armed polymer composition) is typically 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% of the fully loaded value.This corresponds to an average number of D per multi-arm polymerconjugate ranging from about 3.60 to 4.0.

In yet another embodiment, for a multi-armed polymer conjugatecomposition, e.g., where the number of polymer arms ranges from about 3to about 8, the majority (e.g., greater than 50%, but more preferablygreater than 60%, still more preferably greater than 70%, still yet morepreferably greater than 80%, and most preferably greater than 90%) ofspecies present in the composition are those having either an idealizednumber of drug molecules attached to the polymer core (“q”) or thosehaving a combination of (“q”) and (“q−1”) drug molecules attached to thepolymer core.

In accordance with the foregoing, the hydrohalide salt (and compositionscontaining the same) may comprise any one or more of the followingstructures, in addition to the fully drug loaded structure (e.g., havinga glycine-modified irinotecan molecule covalently attached to each ofthe four polymer arms):

For a given polymer arm terminus shown above having a carboxylic acid(and therefore not covalently attached to an active agent, e.g.,irinotecan), other possible termini extending from the 4-arm-PEG-CM(“CM”=—CH₂C(O)—) arm include —OH, —OCH₃,

—NH—CH₂—C(O)—OH, NH—CH₂—C(O)—OCH₃,

For example, provided herein is a composition comprising a plurality of4-armed pentaerythritolyl-tetrapolyethylene glycol-carboxymethylconjugates, wherein at least 90% of the conjugates in the composition(i) have a structure encompassed by the formula:

C—[CH₂—O—(CH₂CH₂O)_(n)—CH₂—C(O)-TERM]₄,

wherein n, in each instance, is an integer having a value from 20 toabout 500, or from 40 to about 500, and TERM, in each instance, isselected from the group consisting of —OH, —OCH₃,

—NH—CH₂—C(O)—OH, —NH—CH₂—C(O)—OCH₃,

and —NH—CH₂—C(O)—O-Irino (“GLY-Irino”), wherein Irino is a residue ofirinotecan; and

-   -   (ii) for each Term in the at least 90% of the four-arm        conjugates in the composition, at least 90% thereof are        —NH—CH₂—C(O)—O-Irino, and (iii) of the at least 90%        —NH—CH₂—C(O)—O-Irinotecan moieties in the composition, at least        90 mole percent of irinotecan's basic nitrogen atoms are        protonated in hydrohalide form such as the hydrochloride salt.        Preferably, of the at least 90%-NH—CH₂—C(O)—O-Irinotecan        moieties in the composition, at least 91 mole percent, or at        least 92 mole percent, or at least 93 mole percent, or at least        94 mole percent or at least 95 mole percent or greater than 95        mole percent of irinotecan's basic nitrogen atoms are protonated        in hydrohalide form, wherein hydrohalide content can be        determined by ion chromatography.

The multi-arm polymer conjugate compositions provided herein areintended to encompass any and all stereoisomeric forms of the conjugatescomprised in such compositions. In a particular embodiment of theconjugate, the stereochemistry at C-20 of irinotecan, when in conjugatedform such as in compositions of 4-arm-PEG-Gly-Irino, remains intact,i.e., C-20 retains its (S)-configuration when in its conjugated form.See, e.g., Example 4.

A preferred multi-armed structure is a carboxymethyl modified 4-armpentaerythritolyl PEG having a glycine linker intervening between thepolymer portion in each arm and the active agent (polymer portion andlinker shown above), where the active agent is7-ethyl-10-hydroxy-camptothecin. Again, included herein are embodimentsin which the multi-arm polymer is (i) fully loaded, as well as having(ii) three 7-ethyl-10-hydroxy-camptothecin molecules covalently attachedthereto, (iii) two 7-ethyl-10-hydroxy-camptothecin molecules covalentlyattached thereto, and (iv) one 7-ethyl-10-hydroxy-camptothecin moleculecovalently attached to the four-arm polymer core. Typical drug loadingsare as previously described.

Yet another representative multi-armed conjugate structure is acarboxymethyl modified 4-arm glycerol dimer (3,3′-oxydipropane-1,2-diol)PEG having 7-ethyl-10-hydroxy-camptothecin (SN-38) molecules covalentlyattached to the polymer core. Embodiments in which the multi-armedpolymer core is fully loaded with drug (i.e., having four7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereo),or is less than fully loaded (i.e., having one, two, or three7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereto)are included herein. The conjugate having drug (i.e.7-ethyl-10-hydroxy-camptothecin) covalently attached to each polymer armis shown below.

In yet another illustrative embodiment, the conjugate is a multi-armedstructure comprising a carboxymethyl modified 4-arm glycerol dimer(3,3′-oxydipropane-1,2-diol) PEG having irinotecan molecules covalentlyattached to the polymer core. Embodiments in which the multi-armedpolymer core is fully loaded with drug (i.e., having four irinotecanmolecules covalently attached thereo), or is less than fully loaded(i.e., having one, two, or three irinotecan molecules covalentlyattached thereto) are included herein.

Parameters of the Hydrohalide Salts

The subject compositions are hydrohalide salts, typically hydrochloridesalts. That is to say, conjugates such as described above are providedin a composition such that at least 90% of basic nitrogen atoms in theconjugate (as well as in the bulk composition) are present in protonatedform (i.e., as the hydrohalide salt). The hydrohalide salt compositionsare stably and reproducibly prepared.

A hydrohalide salt as provided herein is characterized in terms of itsbulk or macro properties. That is to say, basic nitrogen atoms (i.e.amino groups) in the conjugate exist in nearly fully protonated form.While the present compositions are characterized based on bulkproperties, different individual molecular species are typicallycontained within the bulk composition. Taking the exemplary 4-armpolymer conjugate described in Example 6, 4-arm-PEG-Gly-Irino-20Khydrochloride, the salt product contains any of a number of individualmolecular species, although at least 90% overall are protonated as thehydrohalide salt. One molecular species is one in which each polymer armcontains an irinotecan molecule that is in neutral form, i.e., its aminogroup is unprotonated. See structure I below. Another molecular speciesis one in which each polymer arm contains an irinotecan molecule inprotonated form. See structure IV below. An additional molecular speciesis one in which three of the polymer arms contain an irinotecan moleculethat is in protonated form, and one polymer arm contains an irinotecanmolecule in neutral form (structure III). In another molecular species,two of the four polymer arms contain an irinotecan molecule in neutralform (i.e., its amino group is unprotonated), and two of the fourpolymer arms contain an irinotecan molecule that is in protonated form(structure II).

As demonstrated in Example 1, certain polymer prodrug conjugates can beobtained as mixed acid salts of both hydrochloric acid andtrifluoroacetic acid. In Example 1, hydrochloric acid is introduced bythe use of an acid salt form of the active agent molecule to form theresulting polymer conjugate, while the trifluoroacetic acid isintroduced to the reaction mixture in a deprotection step. Followingcovalent attachment of the active agent (or modified active agent asillustrated in Example 1) to the water soluble polymer reagent, andtreatment with base, even in instances in which additional purificationsteps are carried out, the resulting conjugate is obtained as a partialmixed acid salt.

The mixed acid salt conjugates generally contain defined proportions andranges of each component (i.e., free base, inorganic acid salt, organicacid salt). A positive correlation was observed between increasedstability towards hydrolysis and increased molar percentage of salt inthe final conjugate product. Based upon the slopes of the graphs, it canbe determined that as free base content increases, product stabilitydecreases.

FIG. 2 further illustrates that stability (or resistance) againsthydrolytic degradation is greater for conjugates possessing a greaterdegree of protonated amine groups (i.e., acid salt). For instance, itwas observed that conjugate product containing 14 molar percent or morefree base was notably less stable towards hydrolysis than thecorresponding acid salt-rich product.

Additionally, as illustrated in FIG. 3, product rich in thehydrochloride salt appears to be more somewhat more susceptible tocleavage of the poly(ethylene glycol) backbone under accelerated stressconditions than the mixed salt form containing a measurable amount offree base, although buffering in the final composition may be effectiveto ameliorate this feature or tendency.

These collective results indicate the unexpected advantages of ahydrohalide salt of a poly(ethylene glycol)-active agent conjugate (suchas 4-arm-PEG-Gly-Irino-20K) over free base alone.

Hydrohalide Salts Methods for Forming

Upon forming and characterizing the mixed acid salt, a method wasdevised to synthesize a full hydrohalide salt, i.e., one having at least90% of irinotecan's basic nitrogen atoms protonated in hydrohalide saltform as provided in detail in Example 6. An acid salt of a water solublepolymer conjugate can be prepared from commercially available startingmaterials in view of the guidance presented herein, coupled with what isknown in the art of chemical synthesis.

Linear, branched, and multi-arm water-soluble polymer reagents areavailable from a number of commercial sources as described above.Alternatively, PEG reagents, such as a multi-armed reactive PEG polymermay be synthetically prepared as described herein. See, e.g., Example 7herein.

The acid salt can be formed using known chemical coupling techniques forcovalent attachment of activated polymers, such as an activated PEG, toa biologically active agent (See, for example, POLY(ETHYLENE GLYCOL)CHEMISTRY AND BIOLOGICAL APPLICATIONS, American Chemical Society,Washington, D.C. (1997); and U.S. Patent Publication Nos. 2009/0074704and 2006/0239960). Selection of suitable functional groups, linkers,protecting groups, and the like to achieve a mixed acid salt inaccordance with the invention, will depend, in part, on the functionalgroups on the active agent and on the polymer starting material and willbe apparent to one skilled in the art, based upon the content of thepresent disclosure. In view of certain features of the acid salt, themethod comprises provision of an amine (or other basicnitrogen)-containing active agent in the form of an inorganic acidaddition salt, and an inorganic acid treatment step. Reference to a“active agent” in the context of the synthetic method is meant toencompass an active agent optionally modified to possess a linkercovalently attached thereto, to facilitate attachment to thewater-soluble polymer.

Generally, the method comprises the steps of (i) deprotecting aninorganic acid (hydrohalic) salt of an amine-(or other basicnitrogen)-containing active agent in protected form (e.g.,glycine-irinotecan hydrohalide in protected form) by treatment with amolar excess of hydrohalic acid to thereby remove the protecting groupto form a deprotected acid salt such as glycine-irinotecan hydrohalide,(ii) coupling the deprotected inorganic acid salt of step (i) with awater-soluble polymer reagent such as4-arm-pentaerythritolyl-polyethylene glycol-carboxymethyl-succinimide(or a chemically equivalent activated ester or the like), in thepresence of a base to form a polymer-active agent conjugate such as4-arm-pcntaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt (also referredto as pentaerythritolyl-4-arm-(PEG-1-methylene-2-oxo-vinylamino acetatelinked-irinotecan hydrohalide salt), and (iii) recovering thepolymer-active agent conjugate, 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt, byprecipitation.

The resulting polymer-active agent conjugate composition ischaracterized by having at least 90% mole percent of the conjugate'sactive agent's basic amino groups, e.g., irinotecan's basic aminogroups, protonated in hydrohalide salt form. Generally, the molepercentage of hydrohalide groups in the product is determined by ionchromatography.

In turning now to one of the preferred classes of active agents, thecamptothecins, since the 20-hydroxyl group of compounds within thecamptothecin family is sterically hindered, a single step conjugationreaction is difficult to accomplish in significant yields. As a result,a preferred method is to react the 20-hydroxyl group of the bioactivestarting material, e.g., irinotecan hydrochloride, with a short linkeror spacer moiety carrying a functional group suitable for reaction witha water-soluble polymer. Such an approach is applicable to many smallmolecules, particularly those having a site of covalent attachment thatis inaccessible to an incoming reactive polymer. Preferred linkers forreaction with a hydroxyl group to form an ester linkage includet-BOC-glycine or other amino acids such as alanine, glycine, isoleucine,leucine, phenylalanine, and valine having a protected amino group and anavailable carboxylic acid group (See Zalipsky et al., “Attachment ofDrugs to Polyethylene Glycols”, Eur. Polym. J., Vol. 19, No. 12, pp.1177-1183 (1983)). Other spacer or linker moieties having an availablecarboxylic acid group or other functional group reactive with a hydroxylgroup and having a protected amino group can also be used in lieu of theamino acids described above.

Typical labile protecting groups, e.g., for protecting the glycine aminogroup, include t-BOC and FMOC (9-flourenylmethloxycarbonyl). t-BOC isstable at room temperature and easily removed with dilute solutions oftrifluoroacetic acid and dichloromethane. It can also be removed bytreatment with acid, such as an inorganic, hydrohalid acid. FMOC is abase labile protecting group that is easily removed by concentratedsolutions of amines (usually 20-55% piperidine in N-methylpyrrolidone).

In Example 6, directed to the preparation of 4-arm-PEG20K-irinotecanhydrochloride, the carboxyl group of N-protected glycine reacts with the20-hydroxyl group of irinotecan hydrochloride (or other suitablecamptothecin, such as 7-ethyl-10-hydroxy-camptothecin, or any otheractive agent) in the presence of a coupling agent (e.g.,dicyclohexylcarbodiimide (DCC)) and a base catalyst (e.g.,dimethylaminopyridine (DMAP) or other suitable base) to provideN-protected linker modified active agent, e.g., t-Boc-glycine-irinotecanhydrochloride. Although hydrochloride is exemplified, other inorganicacid salts may be used. Preferably, each reaction step is conductedunder an inert, dry atmosphere.

In a subsequent step, the amino protecting group,t-BOC(N-tert-butoxycarbonyl), is removed by treatment with hydrochloricacid (or another hydrohalic acid) under suitable reaction conditions.This differs from the preparation of the mixed acid salt, where t-BOC isremoved by treatment with trifluoroacetic acid as in Example 1. Theresulting deprotected intermediate is linker modified active agent,e.g., 20-glycine-irinotecan hydrochloridel. Illustrative reactionconditions are described in Example 6, and may be further optimized byroutine optimization by one of skill in the art. Generally a molarexcess of acid is used to remove the protecting group. Preferably, theprotected glycine-irinotecan is treated with a ten-fold or greater molarexcess of hydrohalic acid to remove the protecting group. In some cases,a molar excess of 10-fold to 25-fold may be employed. The resultingdeprotected drug salt is typically recovered form the reaction mixture,e.g., by precipitation. As an example, addition of methyltert-butylether (MTBE) may be employed to precipitate the intermediate. Theintermediate product is then isolated, e.g., by filtration, and dried.

Deprotected active agent (optionally linker modified), e.g.,20-glycine-irinotecan HCl, is then coupled to a desired polymer reagent.e.g., 4-arm pentaerythritolyl-PEG-succinimide (or any other similarlyactivated ester counterpart, the nature of which has been previouslydescribed) in the presence of a base (e.g., DMAP, trimethyl amine,triethyl amine, etc.), to form the desired conjugate. The conjugationstep may be conducted in the presence of excess base, e.g., from about1.1 to about 3.0-fold molar excess. Reaction yields for the couplingreaction are typically high, greater than about 90%.

The acid salt conjugate is recovered, e.g., by precipitation with ether(e.g., methyl tert-butyl ether, diethyl ether) or other suitablesolvent. In order to ensure formation of the full hydrohalide salt(i.e., at least 90 mole percent hydrohalide salt), the crude product isanalyzed, e.g., by ion chromatography, to determine halide content. Inthe event that hydrohalide content is less than desired, e.g., less than90 mole percent, or less than 91, 92, 93, 94, or 95 mole percent, theconjugate acid salt is then dissolved in a suitable solvent such asethyl acetate or the like, and treated with additional hydrohalic acid.The product is then recovered as described above.

The acid salt product may be further purified by any suitable method.Methods of purification and isolation include precipitation followed byfiltration and drying, as well as chromatography. Suitablechromatographic methods include gel filtration chromatography, ionexchange chromatography, and Biotage Flash chromatography. Anothermethod of purification is recrystallization. For example, the partialacid salt is dissolved in a suitable single or mixed solvent system(e.g., isopropanol/methanol), and then allowed to crystallize.Recrystallization may be conducted multiple times, and the crystals mayalso be washed with a suitable solvent in which they are insoluble oronly slightly soluble (e.g., methyl tert-butyl ether ormethyl-tert-butyl ether/methanol). The purified product may optionallybe further air or vacuum dried.

Preferably, the acid salt product is stored under conditions suitablefor protecting the product from exposure to any one or more of oxygen,moisture, and light. Any of a number of storage conditions or packagingprotocols can be employed to suitably protect the acid salt productduring storage. In one embodiment, the product is packaged under aninert atmosphere (e.g., argon or nitrogen) by placement in one or morepolyethylene bags, and placed in an aluminum lined polyester heatsealable bag.

Representative mole percents of hydrochloric acid salt is provided inExample 6. As described therein, 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrochloride salt, was preparedas the full hydrochloride salt, i.e., containing nearly 99 mole percentchloride.

Hlydrohalide Salts Pharmaceutical Compositions Containing HydrohalideSalt Conjugates

The acid salt may be in the form of a pharmaceutical formulation orcomposition for either veterinary or human medical use. An illustrativeformulation will typically comprise the acid salt in combination withone or more pharmaceutically acceptable carriers, and optionally anyother therapeutic ingredients, stabilizers, or the like. The carrier(s)must be pharmaceutically acceptable in the sense of being compatiblewith the other ingredients of the formulation and not unduly deleteriousto the recipient/patient. The hydrohalic acid salt is optionallycontained in bulk or in unit dose form in a container or receptaclewhich includes packaging that protects the product from exposure tomoisture and oxygen.

The pharmaceutical composition may include polymericexcipients/additives or carriers, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin. Thecompositions may further include diluents, buffers, binders,disintegrants, thickeners, lubricants, preservatives (includingantioxidants), flavoring agents, taste-masking agents, inorganic salts(e.g., sodium chloride), antimicrobial agents (e.g., benzalkoniumchloride), sweeteners, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80”, and pluronics such asF68 and F88, available from BASF), sorbitan esters, lipids (e.g.,phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g.,cholesterol)), and chelating agents (e.g., EDTA, zinc and other suchsuitable cations). Other pharmaceutical excipients and/or additivessuitable for use in the compositions according to the invention arelisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and in “Handbookof Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The acid salt may be formulated in a composition suitable for oral,rectal, topical, nasal, ophthalmic, or parenteral (includingintraperitoneal, intravenous, subcutaneous, or intramuscular injection)administration. The acid salt composition may conveniently be presentedin unit dosage form and may be prepared by any of the methods well knownin the art of pharmacy. All methods include the step of bringing theacid salt into association with a carrier that constitutes one or moreaccessory ingredients.

In one particular embodiment, the acid salt, e.g.,4-arm-PEG-Gly-Irino-20K hydrohalide is provided in lyophilized form in asterile single use vial for use by injection. In one embodiment, theamount of conjugate product contained in the single use vial is theequivalent of a 100-mg dose of irinotecan. More particularly, thelyophilized composition includes 4-arm-PEG-Gly-Irino-20K hydrohalidesalt combined with lactate buffer at pH 3.5. That is to say, thelyophilized composition is prepared by combining 4-arm-PEG-Gly-Irino-20Khydrohalide, e.g., in an amount equivalent to a 100-mg dose ofirinotecan, with approximately 90 mg of lactic acid, and the pH of thesolution adjusted to 3.5 by addition of either acid or base. Theresulting solution is then lyophilized under sterile conditions, and theresulting powder is stored at −20° C. prior to use. Prior to intravenousinfusion, the lyophilized composition is combined with a solution ofdextrose, e.g., a 5% (w/w) solution of dextrose.

The amount of acid salt (i.e., active agent) in the formulation willvary depending upon the specific active agent employed, its activity,the molecular weight of the conjugate, and other factors such as dosageform, target patient population, and other considerations, and willgenerally be readily determined by one skilled in the art. The amount ofconjugate in the formulation will be that amount necessary to deliver atherapeutically effective amount of the compound, e.g., an alkaloidanticancer agent such as irinotecan or SN-38, to a patient in needthereof to achieve at least one of the therapeutic effects associatedwith the compound, e.g., for treatment of cancer. In practice, this willvary widely depending upon the particular conjugate, its activity, theseverity of the condition to be treated, the patient population, thestability of the formulation, and the like. Compositions will generallycontain anywhere from about 1% by weight to about 99% by weightconjugate, typically from about 2% to about 95% by weight conjugate, andmore typically from about 5% to 85% by weight conjugate, and will alsodepend upon the relative amounts of excipients/additives contained inthe composition. More specifically, the composition will typicallycontain at least about one of the following percentages of conjugate:2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or more by weight.

Compositions suitable for oral administration may be provided asdiscrete units such as capsules, cachets, tablets, lozenges, and thelike, each containing a predetermined amount of the conjugate as apowder or granules; or a suspension in an aqueous liquor or non-aqueousliquid such as a syrup, an elixir, an emulsion, a draught, and the like.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the prodrug conjugate, whichcan be formulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of themulti-armed polymer conjugate with preservative agents and isotonicagents. Such formulations are preferably adjusted to a pH and isotonicstate compatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the multi-armed polymer conjugatedissolved or suspended in one or more media such as mineral oil,petroleum, polyhydroxy alcohols or other bases used for topicalformulations. The addition of other accessory ingredients as noted abovemay be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, e.g., by inhalation. These formulationscomprise a solution or suspension of the desired multi-armed polymerconjugate or a salt thereof. The desired formulation may be placed in asmall chamber and nebulized. Nebulization may be accomplished bycompressed air or by ultrasonic energy to form a plurality of liquiddroplets or solid particles comprising the conjugates or salts thereof.

Hydrohalide Salts Methods of Using the Hydrohalide Salt Conjugates

The acid salt described herein can be used to treat or prevent anycondition responsive to the unmodified active agent in any animal,particularly in mammals, including humans. One representative acid salt,4-arm-pentaerythritolyl-PEG-glycine-irinotecan hydrochloride, comprisingthe anti-cancer agent, irinotecan, is particularly useful in treatingvarious types of cancer.

The acid salt conjugate, in particular, those where the small moleculedrug is an anticancer agent such as a camptothecin compound as describedherein (e.g., irinotecan or 7-ethyl-10-hydroxy-camptothecin) or otheroncolytic, is useful in treating solid type tumors such as breastcancer, ovarian cancer, colon cancer, gastric cancer, malignantmelanoma, small cell lung cancer, non-small cell lung cancer, thyroidcancers, kidney cancer, cancer of the bile duct, brain cancer, cervicalcancer, maxillary sinus cancer, bladder cancer, esophageal cancer,Hodgkin's disease, adrenocortical cancer, and the like. Additionalcancers treatable with the acid salt include lymphomas, leukemias,rhabdomyosarcoma, neuroblastoma, and the like. As stated above, thesubject conjugate is particularly effective in targeting andaccumulating in solid tumors. The conjugate is also useful in thetreatment of HIV and other viruses.

Representative conjugates such as4-arm-pentaerythritolyl-PEG-glycine-irinotecan have also been shown tobe particularly advantageous when used to treat patients having cancersshown to be refractory to treatment with one or more anticancer agents.

Methods of treatment comprise administering to a mammal in need thereofa therapeutically effective amount of an acid salt composition orformulation as described herein.

Additional methods include treatment of (i) metastatic breast cancerthat is resistant to anthracycline and/or taxane based therapies, (ii)platinum-resistant ovarian cancer, (iii) metastatic cervical cancer, and(iv) colorectal cancer in patients with K-Ras mutated gene status byadministering an acid salt composition as described herein.

In treating metastatic breast cancer, an acid salt of a conjugate suchas 4-arm-pentaerythritolyl-PEG-glycine-irinotecan as provided herein isadministered to a patient with locally advanced metastatic breast cancerat a therapeutically effective amount, where the patient has had no morethan two prior (unsuccessful) treatments with anthracycline and/ortaxane based chemotherapeutics.

For treating platinum-resistant ovarian cancer, a composition asprovided herein is administered to a patient with locally advanced ormetastatic ovarian cancer at a therapeutically effective amount, wherethe patient has shown tumor progression during platinum-based therapy,with a progression-free interval of less than six months.

In yet another approach, a hydrohalic acid salt (e.g., such as that inExample 6) is administered to a subject with locally advanced colorectalcancer, where the colorectal tumor(s) has a K-Ras oncogene mutation(K-Ras mutant types) such that the tumor does not respond toEGFR-inhibitors, such as cetuximab. Subjects are those having failed oneprior 5-FU containing therapy, and are also irinotecan naïve.

A therapeutically effective dosage amount of any specific acid salt willvary from conjugate to conjugate, patient to patient, and will dependupon factors such as the condition of the patient, the activity of theparticular active agent employed, the type of cancer, and the route ofdelivery.

For camptothecin-type active agents such as irinotecan or7-ethyl-10-hydroxy-camptothecin, dosages from about 0.5 to about 100 mgcamptothecin/kg body weight, preferably from about 10.0 to about 60mg/kg, are preferred. When administered conjointly with otherpharmaceutically active agents, even less of the acid salt may betherapeutically effective. For administration of an acid salt ofirinotecan as exemplified herein, the dosage amount of irinotecan willtypically range from about 50 mg/m² to about 350 mg/m².

Methods of treatment also include administering a therapeuticallyeffective amount of an cid salt composition or formulation as describedherein (e.g., where the active agent is a camptothecin type molecule) inconjunction with a second anticancer agent. Preferably, suchcamptothecin-based conjugates in the form of an acid salt, areadministered in combination with 5-fluorouracil and folinic acid asdescribed in U.S. Pat. No. 6,403,569.

The hydrohalic acid salt compositions may be administered once orseveral times a day, preferably once a day or less. The duration of thetreatment may be once per day for a period of from two to three weeksand may continue for a period of months or even years. The daily dosecan be administered either by a single dose in the form of an individualdosage unit or several smaller dosage units or by multipleadministration of subdivided dosages at certain intervals.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully described in theliterature. Reagents and materials are commercially available unlessspecifically stated to the contrary. See, for example, M. B. Smith andJ. March, March's Advanced Organic Chemistry: Reactions Mechanisms andStructure, 6th Ed. (New York: Wiley-Interscience, 2007), supra, andComprehensive Organic Functional Group Transformations II, Volumes 1-7,Second Ed.: A Comprehensive Review of the Synthetic Literature 1995-2003(Organic Chemistry Series), Eds. Katritsky, A. R., et al., ElsevierScience.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

The following examples illustrate certain aspects and advantages of thepresent invention, however, the present invention is in no wayconsidered to be limited to the particular embodiments described below.

ABBREVIATIONS Ar argon CM carboxymethyl or carboxymethylene (—CH₂COOH)DCC 1,3-dicyclohexylcarbodiimide DCM dichloromethane DMAP4-(N,N-dimethylamino)pyridine GLY glycine HCl hydrochloric acid RP-HPLCreverse-phase high performance liquid chromatography IPA isopropylalcohol IRT irinotecan IPC ion pair chromatography MeOH methanol MTBEmethyl tert-butyl ether MW molecular weight NMR nuclear magneticresonance PEG polyethylene glycol RT room temperature SCMsuccinimidylcarboxymethyl (—CH₂—COO—N— succinimidyl) TEA triethylamineTFA trifluoroacetic acid THF tetrahydrofuran

Materials and Methods

Pentaerythritolyl-based 4-ARM-PEG_(20K)-OH was obtained from NOFCorporation (Japan). 4-ARM-PEG_(20K)-OH possesses the structure:C—(CH₂O—(CH₂CH₂O)_(N)H)₄, wherein each n is about 113.

Additional suppliers of pentaerythritolyl-based 4-ARM-PEG_(20K)-OH (alsoreferred to simply as 4-Arm PEG-OH) include Creative PEGWorks(Winston-Salem, N.C.), which also offers the succinimidyl-functionalizedversion, and JenKem Technology USA (Allen, Tex.).

All ¹HNMR data was generated by a 300 or 400 MHz NMR spectrometermanufactured by Bruker.

Example 1 Preparation ofPentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino AcetateLinked-Irinotecan)-20K “4-Arm-PEG-Gly-Irino-20K” Mixed TrifluoroaceticAcid.Hydrochloride Salt

Reaction Scheme:

This example describes the synthesis of a mixed TFA.HCl acid salt of4-Arm-PEG-Gly-Irino-20K.

All solvents used in the synthesis were anhydrous.

Step 1. Conjugation of t-boc-glycine to Irinotecan.HCl Salt (>95% Yield)

Irinotecan.HCl.trihydrate (1 mole or 677 g) and DMF (10 L) were chargedinto a distiller at 60° C. Upon dissolution of theirinotecan.HCl.trihydrate in DMF, full vacuum was slowly applied inorder to remove water from the irinotecan.HCl.trihydrate by azeotropicdistillation at 60° C. Upon solids formation from the residual DMF,heptane (up to 60 L) was charged into the distiller to remove residualDMF at 40-50° C. Upon removal of heptane by visual inspection, theazeotropic distillation was stopped and the solid (irinotecan.HCl) wasallowed to cool to 17±2° C. For the coupling reaction, t-boc-glycine(1.2 mole), 4-DMAP (0.1 mole) dissolved in DCM (1 L), and DCM (19 L)were charged into the distiller. Once the mixture was visually welldispersed, melted DCC (1.5 mole) was added and reaction was allowed toproceed. The reaction was carried out under an argon or nitrogenblanket, with sufficient mixing and pot temperature at 17±2° C.

After a 2-4 hour reaction time, a sample was withdrawn to measureresidual irinotecan (IRT) peak area percent by chromatography. Residualirinotecan was determined to be present in an amount of no more than 5%.DCU formed during the coupling reaction was removed by filtration, andwashed with DCM. The resulting filtrates containing crudet-boc-glycine-irinotecan.HCl salt were combined and concentrated below45° C. under vacuum to remove DCM. When approximately 75% of its initialvolume was removed by distillation. IPA was then added to theconcentrate to reach the initial volume, and the mixture furtherdistilled until the condensate volume reached about 25% of its initialvolume. The resulting clear solution was cooled to room temperature,followed by its addition to heptane with mixing. The mixture was mixedfor an additional 0.5 to 1 hour, during which time a precipitate formed.The precipitate was drained and filtered to obtain a wet cake, and thenwashed with heptane (up to 6 L). The wet cake was vacuum-dried to yieldt-boc-glycine-irinotecan powder for use in Step 2. Yield >95%.

Step 2. Deprotection of t-boc-glycine-Irinotecan

The t-boc-glycine-irinotecan (1 mole) from Step 1 was dissolved in DCMwith agitation to form a visually homogeneous solution. To this solutionwas added TFA (15.8 mole) over a period of 5 to 10 minutes and theresulting solution stirred for about 2 hours. Residual starting materialwas measured by RP-HPLC and determined to be less than about 5%.Acetonitrile was then added to the reaction solution to form a visuallyhomogeneous solution at RT. This solution was then added to MTBE (46.8kg) being sufficiently agitated at 35° C. to promote crystallization.Optionally to reduce MTBE use, DCM in the reaction solution was replacedwith acetonitrile by distillation at 15 to 40° C. After the solventswap, the product-containing solution was added into approximately 50%less volume of MTBE (23 kg) being sufficiently agitated at thecrystallization temperature (35° C.). Mixing was continued for a half toone hour. The resulting solid was filtered and the cake washed withMTBE.

The wet cake was vacuum-dried to yield the glycine-irinotecan saltpowder for use in Step 3. Trifluoroacetate and chloride content of theproduct was determined by ion chromatography with a conductivitydetector. (Yield >95%).

Step 3. PEGylation of Glycine-Irinotecan Using 4-arm-PEG-CM-SCM

The glycine-irinotecan.TFA/HCl salt powder from Step 2 was added to areaction vessel to which was added DCM (approx. 23 L). The mixture wasagitated for approximately 10 to 30 minutes to allow theglycine-irinotecan-TFA/HCl salt to disperse in DCM. Triethyl amine(approx. 1.05 moles (HCl+TFA) moles in glycine-irinotecan TFA/HCl saltpowder) was then added slowly, at a rate which maintained the pottemperature at 24° C. or below. The resulting mixture was agitated for10 to 30 minutes to allow dissolution of the GLY-IRT (glycine-modifiedirinotecan) free base.

Approximately 80% of the total quantity (6.4 kg) of 4-arm PEG-SCM-20kDwas added to the reaction vessel over a course of up to 30 minutes.After dissolution of the PEG reagent, reaction progress was monitored byIPC. (In the event that the amount of non-conjugated GLY-IRT was greaterthan 5% when the reaction appeared to have reached a plateau, theremaining 20% of 4-arm PEG SCM was then added to the reaction vessel,and the reaction progress monitored until a constant value of unreactedGLY-IRT was observed).

Crude product was precipitated by adding the reaction solution into MTBE(113.6 L) agitated at room temperature over a period of from 1-1.5hours, followed by stirring. The resulting mixture was transferred intoa filter-drier with an agitator to remove the mother liquor. Theprecipitate (crude product) was partially vacuum-dried at approximatelyat 10 to 25° C. with minimum intermittent stirring.

Crude product was then placed into a reaction vessel, to which was addedIPA (72 L) and MeOH (8 L), followed by agitation for up to 30 minutes.Heat was applied to achieve visually complete dissolution (a clearsolution) at 50° C. pot temperature, followed by agitation for 30 to 60minutes. The solution was then cooled to 37° C., held there for severalhours, followed by cooling to 20° C. The mixture was transferred into anagitated filter dryer, and filtered to remove mother liquor to form acake on a filter. The cake was washed with 70% MTBE in IPA and 30% MeOHand partially vacuum-dried. This procedure was repeated two additionaltimes, with the exception that, prior to cooling, the clear IPA/MeOHsolution containing 4-arm PEG-Gly-IRT was filtered using an in-linefilter (1 um) at 50° C. to remove any potential particulates in the last(3rd) crystallization.

Three representative samples were taken from the washed wet cake, andNHS levels were measured using NMR. The wet cake was vacuum-dried.

The product (“API”) was packaged into double bags sealed under an inertatmosphere, and stored at −20° C. without exposure to light. Productyield was approximately 95%.

Example 2

Characterization of “4-Arm-PEG-Gly-Irino-20K” Product from Example 1 asa Mixed Salt

The product from Example 1 was analyzed by ion chromatography (ICanalysis). See Table 1 below for IC analytical results for variousproduct lots of 4-arm-PEG-Gly-Irino-20K.

TABLE 1 Mole Percent of Irinotecan bound to PEG LOT NO. TFA SALT HClSALT FREE BASE 010 59 36  5 (low) 020 64 (high) 30 6 030 27 (low)  24 49(high) 040 53 26 21 050 54 26 20 060 57 28 15 070 53 33 14 080 53 27 20090 44 19 36 100 33 41 26 Average of last 50 29 22 7 lots

Based upon the IC results provided in Table 1, it can be seen that theproduct formed in Example 1, 4-arm-PEG-Gly-Irino-20K, is a partial mixedsalt of approximately 50 mole percent TFA salt, 30 mole percent HClsalt, and 20 mole percent free base, based upon conjugated irinotecanmolecules in the product. The mixture of salts was observed even afterrepeated (1-3) recrystallizations of the product. In the various productlots analyzed above, it can be seen that about 35-65 mole percent of theirinotecan molecules in the composition are protonated as the TFA salt,about 25-40 mole percent of the irinotecan molecules in the compositionare protonated as the HCl salt, while the remaining 5-35 mole percent ofthe irinotecan is non-protonated (i.e., as the free base).

The generalized structure of the product is shown below, where theirinotecan moieties are shown in free base form, and in association withHCl and TFA—as an indication of the mixed salt nature of the product.

Example 3 Stress Stability Studies of 4-Arm-PEG-Gly-Irino-20K

Accelerated stability studies were conducted in an attempt to evaluatethe 4-arm-PEG-Gly-Irino-20K product composition. Compositions containingvarying amounts of protonated irinotecan, as well as differing in theamount of TFA versus HCl salt were examined.

Stress Stability Studies

The product formed in Example 1, 4-arm-PEG-Gly-Irino-20K, compound 5,(approximately 1-2 g) was weighed into PEG PE ‘whirl top’ bags andplaced into another ‘whirl top’ bag in order to simulate the APIpackaging conditions. In one study (results shown in FIG. 1), sampleswere placed in an environmental chamber at 25° C./60% RH for 4 weeks. Inanother study, samples were placed in an environmental chamber at 40°C./75% RH for up to several months (results shown in FIG. 2 and FIG. 3).Samples were taken and analyzed on a periodic basis over the course ofthe studies.

Results

The results of the studies are shown in FIG. 1, FIG. 2 and FIG. 3. InFIG. 1, 4-arm-PEG-Gly-Irino-20K peak area percents for samples stored at25° C. and 60% relative humidity are plotted versus time. The data shownare for samples consisting of >99% HCl salt (<1% free base, triangles),94% total salt (6% free base, squares), and 52% total salt (48% freebase, circles). The slopes of the graphs indicate that as free basecontent increases, the stability of the product decreases. Under thestress conditions employed (i.e., 25° C. for up to 28 days), the drop in4-arm-PEG-Gly-Irino-20K peak area correlated well with the increase infree irinotecan, indicating that the mode of decomposition is primarilyvia hydrolysis of the ester bond to release irinotecan. Based upon theresults observed, it appears that a greater amount of free base in theproduct leads to decreased stability towards hydrolysis. Thus, productcontaining a greater degree of protonated irinotecan appears to have agreater stability against hydrolysis than product containing lessprotonated irinotecan (based upon mole percent).

FIG. 2 and FIG. 3 show another set of data obtained from the samplecontaining >99% HCl salt (<1% free base, squares) and a sampleconsisting of 86% total salts (14% free base, diamonds) that were storedat 40° C. and 75% relative humidity. FIG. 2 shows the increase in freeirinotecan over 3 months for both samples. This data is consistent withthe data from the previously described study (summarized in FIG. 1),which shows that product with a higher free base content is less stablewith respect to hydrolysis. FIG. 3 shows the increase in smaller PEGspecies for the same samples over 3 months. The increase in smaller PEGspecies is indicative of decomposition of the PEG backbone to providemultiple PEG species. The data indicates that product corresponding tothe HCl salt is more prone to PEG backbone decomposition than the mixedsalt sample containing 14% free base under accelerated stabilityconditions. Thus, while not intending to be bound by theory, it appearsthat that while the partial mixed salt may degrade primarily byhydrolytic release of drug, the hydrochloride salt appears to degrade bya different mechanism, i.e., degradation of the polymer backbone.However, the extent of backbone degradation can be minimized, e.g., bycontrolling storage conditions.

In summary, the two modes of decomposition observed exhibit oppositetrends with respect to salt/free base content. Although thehydrochloride salt did demonstrate a greater degree of backbonedegradation under accelerated stability testing (possibly due to theacidity of the formulation), the hydrochloride salt was shown to havegreater hydrolytic stability than either the free base or mixedTFA.hydrochloride salt.

Example 4 Chirality Study

The chirality of carbon-20 of irinotecan in 4-arm-PEG-Gly-Irino-20K wasdetermined.

As detailed in documentation from the vendor, the irinotecanhydrochloride starting material is optically active, with C-20 in its(S)-configuration. The C-20 position in irinotecan bears a tertiaryalcohol, which is not readily ionizable, hence this site is not expectedto racemize except under extreme (strongly acidic) conditions. Toconfirm the chirality at the C-20 in 4-arm-PEG-Gly-Irino-20K, a chiralHPLC method was used to analyze irinotecan released from product viachemical hydrolysis.

Based upon the resulting chromatograms, no (R)-enantiomer was detectedfor the 4-arm-PEG-Gly-Irino-20K samples. Following hydrolysis, theirinotecan released from the conjugate was confirmed to be the(S)-configuration.

Example 5 Hydrolysis Study

All PEGylated irinotecan species are considered as part of4-arm-PEG-Gly-Irino-20K (regardless of the particular form-free base,mixed TFA.chloride salt, or chloride salt); each species cleanlyhydrolyzes to produce irinotecan of >99% purity. Furthermore, the main,fully drug-loaded DS4 species (irinotecan covalently attached on each ofthe four polymer arms) and the partially substituted species—DS3(irinotecan covalently attached on three polymer arms), DS2 (irinotecancovalently attached on two of the polymer arms) and DS1 species(irinotecan covalently attached on a single polymer arm)—all hydrolyzeat the same rate to release free drug, irinotecan.

Experiments were performed to determine the fate of theirinotecan-containing PEG species in 4-arm-PEG-Gly-Irino-20K mixedTFA.chloride salt under transesterification (K₂CO₃ in CH₃OH, 20° C.) andaqueous hydrolysis (pH 10, 20° C.) conditions. The transesterificationreaction was >99% complete after 45 minutes. The aqueous hydrolysisreaction was >99% complete within 24 hours. For both reaction types,control reactions using irinotecan were performed under identicalconditions and some artifact peaks were observed. After adjustment forartifact peaks, in both cases, the irinotecans produced hadchromatographic purities of >99%.

Based upon these results, it was concluded that essentially allPEGylated species in 4-arm-PEG-Gly-Irino-20K such as the hydrochloridesalt release irinotecan. Overlays of the HPLCs taken over time from theaqueous hydrolysis reaction show the conversion of DS4 to DS3 to DS2 toDS1 to irinotecan. All of these species hydrolyze to release irinotecanas illustrated in FIG. 4 which demonstrates release of irinotecan viahydrolysis from mono-, di-, tri- and tetra-substituted4-arm-PEG-Gly-Irino-20K species.

Additional experiments were conducted to measure the rates of hydrolysisfor the major component of 4-arm-PEG-Gly-Irino-20K, DS4, and its lessersubstituted intermediates, DS3, DS2 and DS1 in aqueous buffer (pH 8.4)in the presence of porcine carboxypeptidase B and in human plasma. Thehydrolysis in aqueous buffer (pH 8.4) in the presence of porcinecarboxypeptidase B was an attempt to perform enzyme-based hydrolysis.The control experiment at pH 8.4 without the enzyme later showed thatthe hydrolysis was pH-driven, and thus primarily a chemical hydrolysis.The data were, nevertheless, valuable for comparison with the dataobtained from the hydrolysis performed in human plasma. Theseexperiments showed that the hydrolysis rates of the various componentsare not significantly different and compare favorably with theoreticalpredictions. Additional experiments measured the rates of hydrolysis forthe major components (DS4, DS3, DS2 and DS1) of 4-arm-PEG-Gly-Irino-20Kin human plasma. These experiments also show that the various componentsare hydrolyzed at the same rate and compare favorably with theoreticalpredictions.

FIG. 5 and FIG. 6 present graphs which show the theoretical hydrolysisrates versus experimental data for the chemical hydrolysis (in thepresence of enzyme) and plasma hydrolysis, respectively. In both cases,the theoretical predictions are based on identical rates for thehydrolysis of each species to produce the next-lower homologue plus freeirinotecan (i.e., DS4>DS3>DS2>DS1).

Example 6 Preparation ofPentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino AcetateLinked-Irinotecan)-20K “4-Arm-PEG-Gly-Irino 20K” Hydrochloride Salt

Step 1. Synthesis of Boc-Glycine-irinotecan Hydrochloride (Gly-IRT HCl)Part 1: Drying of Irinotecan Hydrochloride Trihydrate (IRT.HCl.3H2O)

IRT.HCl.3H₂O (45.05 g, 66.52 mmol) was charged into a reactor. AnhydrousN,N-dimethylformamide (DMF) (666 mL, 14.7 mL/g of IRT.HCl.3H₂O, DMFwater content no more than 300 ppm) was charged to the reactor. Withslow agitation, the reactor was heated to 60° C. (jacket temperature).After the irinotecan (IRT) was fully dissolved (5-10 minutes) vacuum wasslowly applied to reach 5-10 mbar and the DMF was distilled off. Whenthe volume of condensed distillate (DMF) reached 85-90% of the initialDMF charge, the vacuum was released. Heptane (1330 mL, 30.0 mL/g ofIRT.HCl.3H₂O, water content no more than 50 ppm) was introduced into thereactor and the jacket temperature was lowered to 50° C. Heptane wasvacuum distilled (100-150 mbar) until the volume of the distillate wasabout 90% of the initial charge of heptane. Two more cycles of heptanedistillation were carried out (2×1330 mL heptanes charges anddistillations). A solvent phase sample was removed from the reactor andwas analyzed for DMF content using gas chromatography to ensure that theDMF content of the sample was no more than 3% w/w. (In the event theresidual DMF is >3.0% w/w, a fourth azeotropic distillation cycle isperformed). The resultant slurry was used for the coupling reaction.

Part 2: Coupling Reaction

Dichloromethane (1330 mL, 29.5 mL DCM/g IRT.HCl.3H₂O) was charged intothe reactor where the slurry of dry IRT.HCl (1.0 equiv) in residualheptanes (approximate mass ratio of residual heptane to IRT.HCl was 3)was being stirred. The reaction contents were agitated for 15-30minutes, and the batch temperature was maintained at 17° C. Boc-glycine(14.0 g, 79.91 mmol, 1.2 equiv) and DMAP (0.81 g, 6.63 mmol, 0.1 equiv)were charged, as solids, into the reactor. A DCM solution of DCC (1.5equiv in 40 mL of dichloromethane) was prepared and added over 15-30min, and the resultant reaction mixture was stirred at 17° C. (batchtemperature) for 2-3 hr. The reaction was monitored by HPLC forcompletion. A pre-made quenching solution was charged into the reactionmixture to quench any remaining DCC. Briefly, the pre-made quenchingsolution is a pre-mixed solution of TFA and IPA in dichloromethane,prepared by mixing TFA (1.53 mL, 0.034 mL/g IRT.HCl.3H₂O) and IPA (3.05mL, 0.068 mL/g IRT.HCl.3H₂O) in DCM (15.3 mL, 0.34 mL/g IRT.HCl.3H₂O),and was added to the reactor V1 over 5-10 minutes when the conversionwas at least 97%. The contents were agitated for additional 30-60 min toallow quenching. The DCU-containing reaction mixture was filteredthrough a 1 micron filter into another reactor. The reaction filtratewas distilled to ⅓ volume under vacuum at 35° C. Isopropyl alcohol (IPA)(490.5 mL, 10.9 mL/g IRT.HCl.3H₂O) was added to the concentrated mixtureand the mixture was stirred for 30-60 min at 50° C. (jackettemperature). The resulting homogeneous solution was concentrated byvacuum distillation to approximately 85% of the initial IPA chargevolume and the resultant concentrate was cooled to 20° C. (jackettemperature). The reaction mixture in IPA was transferred over 60-80 mininto heptane (1750 mL, 38.8 mL heptane/g IRT.HCl.3H₂O) at 20° C. Theresultant slurry containing Boc-gly-IRT HCl precipitate was stirred foran additional 60-90 minutes and the product was collected by filtration.The reaction flask was rinsed with heptane (2×490 mL, 20.0 mL Heptane/gIRT.HCl.3H2O) and the product cake was washed with the rinse. The wetcake was dried at 20° C. to 25° C. under vacuum for a minimum of 12 hrs.Yield: 57.13 g (110%, high because of residual solvents).

Step 2. Synthesis of Glycine-Irinotecan Hydrochloride (Gly-IRT HCl)

A 100 ml round bottom flask was charged with BOC-Gly-IRT (2.34 g, 0.003moles) and IPA (12 ml), to which was added HCl (12 ml, 4M, in dioxane,0.045 moles over a period of 10 minutes. The reaction mixture wasstirred at R.T. for 6 hrs (and monitored by HPLC for completion ofreaction), followed by addition of dry acetonitrile (12 mL). Theresulting reaction mixture was slowly added (5 mins) to a stirringsolution of MTBE (140 ml). The solid thus obtained was filtered anddried under vacuum to give Gly-IRT HCl salt as a yellow colored powder.Yield: 2.17 g.

Step 3. Synthesis of 4-arm PEG20K-glycine Irinotecan Hydrochloride

Gly-IRT HCl (5.04 g 14.61 wt % HCl) was charged to a 100 mL reactor andflushed with argon. The jacket temperature was set at 20° C. DCM (100mL) and TEA (4 mL) were added. The solution was stirred for 10 minutes.

An initial charge of 4-arm-PEG20K-SCM was added (26.5 g) and thereaction mixture stirred for 30 minutes. A sample was taken and analyzedvia HPLC. The HPLC data showed 6.1% remaining Gly-IRT.HCl. A secondcharge of 4-arm-PEG20K-SCM (0.68 g) was added to the reaction mixtureand the solution stirred for approximately 2 hours. A sample was takenfor HPLC analysis. The HPLC analysis data showed 1.2% remaining Gly-IRTHCl.

The reaction solution was then slowly added to MTBE (800 mL) toprecipitate the product. The precipitate was stirred for 30 minutes andcollected via filtration. The wet cake was washed with MTBE (200 mL)twice. The product was dried under vacuum. The crude4-arm-PEG20K-irinotecan hydrochloride intermediate was analyzed by ionchromatography for chloride content.

Table 2 summarizes the resulting 4-arm-PEG20K-irinotecan hydrochlorideintermediate salt content analysis (IC chromatography).

TABLE 2 Chloride IRT content Chloride Wt % Wt % Mole % 0.493% 9.8% 83.3%

Salt Adjustment and Ethyl Acetate Isolation:

The crude 4-arm-PEG20K-irinotecan hydrochloride intermediate (29.1 g,83.3 mole % Cl) was dissolved in 600 mL of ethyl acetate at 35° C. Thesolution was stirred for 15 minutes following visible dissolution ofsolids. A 0.1N solution of HCl in ethanol (8.5 ml) was charged to thesolution and stirred for 30 minutes. The flask was immersed in an icebatch with strong stirring. Visible solids precipitated from solutionafter 10 minutes. The mixture was stirred for a total of 60 minutes inthe bath. The precipitate was collected by filtration in a glass frit bythe application of light vacuum. The wet cake was washed with a 30%MeOH/70% MTBE solution (400 ml). The product was placed under vacuum todry. Yield: 28.3 g

The chloride content of the final product (as determined by ionchromatography) was as follows:

IRT Content, Wt % Chloride, Mole % 9.8% 98.8%

Another lot prepared by the above process was determined by ionchromatography to possess 103.8% mole % chloride (i.e., was fully in theform of the hydrochloride salt). When stored and evaluated over a periodof 4 weeks at 40° C., the total product related species changed from98.7% to 97.0%, while free irinotecan changed from 0.4% to 1.25%,indicating the stability of the hydrochloride salt (i.e., resistance)with respect to hydrolytic degradation. Under these same conditions,polyethylene glycol backbone cleavage was detected after 4 weeks but wasnot measurable.

Example 7 Preparation of Pentaerythitol-Based 4-ARM-PEG-20K at 1.9 kgScale

Materials and Methods. A very high grade of ethylene oxide having thelowest water content achievable should be used as water content leads topolymeric diol impurities. CAUTION: Ethylene oxide is a very reactivecompound that can react explosively with moisture, thus leaks in thereaction and transfer apparatus should carefully avoided. Also, careshould be taken in operations to include having personnel work behindprotective shields or in bunkers.

Anhydrous toluene (4 L) was refluxed for two hours in a two gallonjacketed stainless steel pressure reactor. Next, a part of the solvent(3 L) was distilled off under atmospheric pressure. The residual toluenewas then discharged out and the reactor was dried overnight by passingsteam through the reactor jacket and applying reduced pressure 3-5 mmHg. Next the reactor was cooled to room temperature, filled withanhydrous toluene (4 L) and pentaerythitol based 4ARM-PEG-2K (SUNBRIGHTPTE®-2000 pentacrythritol, molecular weight of about 2,000 Daltons, NOFCorporation; 200 g, 0.100 moles) was added. The solvent was distilledoff under reduced pressure, and then the reactor was cooled to 30° C.under dry nitrogen atmosphere. One liter of molecular sieves-driedtoluene (water content˜5 ppm) and liquid sodium-potassium alloy (Na 22%,K 78%; 1.2 g) were added to the reactor. The reactor was warmed to 110°C. and ethylene oxide (1,800 g) was continuously added over three hourskeeping the reaction temperature at 110-120° C. Next, the contents ofthe reactor were heated for two hours at ˜100° C., and then thetemperature was lowered to ˜70° C. Excess ethylene oxide and toluenewere distilled off under reduced pressure. After distillation, thecontents of the reactor remained under reduced pressure and a nitrogensparge was performed to remove traces of ethylene oxide. Phosphoric acid(1N) was added to neutralize the basic residue and the product was driedunder reduced pressure. Finally the product was drained from the reactorand filtered giving after cooling 1,900 g of white solid. Gel FiltrationChromatography (GFC) was applied to characterize the alkoxylatedpolymeric product, pentaerythitol based 4-ARM-PEG-20K. This analyticalmethod provided a chromatogram of the composition with separation of thecomponents according to molecular weight. An Agilent 1100 HPLC systemequipped with Shodex KW-803 GFC column (300×8 mm) and differentialrefractometer detector was used. The flow of the mobile phase (0.1MNaNO₃) was 0.5 ml/min. The GFC chromatogram is shown in FIG. 7.

GFC analysis showed that the 4ARM-PEG-20K product contained thefollowing: High MW product 0.42%, 4ARM-PEG-20K 99.14%, HO-PEG(10K)-OH0.44%.

Example 9 Analysis of Commercially Available 4ARM-PEG-20K

NOF Corporation is a current leader in providing commercial PEGs. Thus afresh commercially available pentaerythritol-based 4ARM-PEG-20K(SUNBRIGHT PTE®-20,000, molecular weight of about 20,000 Daltons, NOFCorporation) was obtained and analyzed using Gel FiltrationChromatography (GFC). An Agilent 1100 HPLC system equipped with ShodexKW-803 GFC column (300×8 mm) and differential refractometer detector wasused. The flow of the mobile phase (0.1M NaNO₃) was 0.5 ml/min. The GFCchromatogram is shown in FIG. 8.

GFC analysis showed that this commercial 4ARM-PEG-20K product contained:High MW products 3.93%, 4ARM-PEG-20K 88.56%, HO-PEG(10K)-OH 3.93%,HO-PEG(5K)-OH 3.58%.

Example 10 Preparation of Alkoxylatable Oligomer Pentaerythritol-Based4-ARM-PEG-2K at 15 Kg Scale

A twenty gallon jacketed stainless steel pressure reactor was washed twotimes with 95 kg of deionized water at 95° C. The wash water was removedand the reactor was dried overnight by passing steam through the reactorjacket and applying reduced pressure (3-5 mm Hg). The reactor was filledwith 25 kg of anhydrous toluene and a part of the solvent was distilledoff under reduced pressure. The residual toluene was then discharged outand the reactor was kept under reduced pressure. Next the reactor wascooled to room temperature, filled with anhydrous toluene (15 L) andpentaerythritol (1,020 g) was added. Part of the solvent (˜8 L) wasdistilled off under reduced pressure, and then the reactor was cooled to30° C. under dry nitrogen atmosphere. Liquid sodium-potassium alloy (Na22%, K 78%; 2.2 g) was added to the reactor. Anhydrous ethylene oxide(14,080 g) was continuously added over three hours keeping the reactiontemperature at 150-155° C. Next, the contents of the reactor were heatedfor 30 min at ˜150° C., and then the temperature was lowered to ˜70° C.Excess ethylene oxide and toluene were distilled off under reducedpressure. After distillation, the contents of the reactor remained underreduced pressure and a nitrogen sparge was performed to remove traces ofethylene oxide. Finally the product was drained from the reactor giving14,200 g of viscous liquid. Gel Filtration Chromatography (GFC) wasapplied to characterize the product, pentaerythritol based 4-ARM-PEG-2K.This analytical method provided a chromatogram of the composition withseparation of the components according to molecular weight. An Agilent1100 HPLC system equipped with Shodex KW-803 GFC column (300×8 mm) anddifferential refractometer detector was used. The flow of the mobilephase (0.1M NaNO₃) was 0.5 ml/min.

GFC analysis showed that the 4ARM-PEG-2K product was ˜100% pure with lowor high molecular weight impurities below detectable limits.

Example 11 Preparation of Pentaerythritol-Based 4-ARM-PEG-20K at 20 KgScale

A twenty gallon jacketed stainless steel pressure reactor was washed twotimes with 95 kg of deionized water at 95° C. Water was discharged outand the reactor was dried overnight by passing steam through the reactorjacket and applying reduced pressure 3-5 mm Hg. The reactor was filledwith 25 kg of toluene and a part of the solvent was distilled off underreduced pressure. The residual toluene was then discharged out and thereactor was kept under reduced pressure. Next the reactor was cooled toroom temperature, filled with anhydrous toluene (21 L) and previouslyisolated alkoxylatable oligomer: pentaerythritol based 4ARM-PEG-2K fromthe Example 10 (2,064 g) was added. Part of the solvent (16 L) wasdistilled off under reduced pressure, and then the reactor was cooled to30° C. under dry nitrogen atmosphere. Four liter of molecularsieves-dried toluene (water content ˜5 ppm) and liquid sodium-potassiumalloy (Na 22%, K 78%; 1.7 g) were added, and the reactor was warmed to110° C. Next ethylene oxide (19,300 g) was continuously added over fivehours keeping the reaction temperature at 145-150° C. Next, the contentsof the reactor were heated for 30 min at ˜140° C., and then thetemperature was lowered to ˜100° C. Glacial acidic acid (100 g) wasadded to neutralize the catalyst. Excess ethylene oxide and toluene weredistilled off under reduced pressure. After distillation, the contentsof the reactor remained under reduced pressure and a nitrogen sparge wasperformed to remove traces of ethylene oxide. Finally the product wasdrained from the reactor giving 20,100 g of white solid. Gel FiltrationChromatography (GFC) was applied to characterize the alkoxylated polymerproduct, pentaerythritol based 4-ARM-PEG-20K. This analytical methodprovided a chromatogram of the composition with separation of thecomponents according to molecular weight. An Agilent 1100 HPLC systemequipped with Shodex KW-803 GFC column (300×8 mm) and differentialrefractometer detector was used. The flow of the mobile phase (0.1 MNaNO₃) was 0.5 ml/min.

GFC analysis showed that the 4ARM-PEG-20K product contained thefollowing: High MW product 0.75%, 4ARM-PEG-20K 97.92%, HO-PEG(10K)-OH1.08%. HO-PEG(5K)-OH 0.48%.

The invention(s) set forth herein has been described with respect toparticular exemplified embodiments. However, the foregoing descriptionis not intended to limit the invention to the exemplified embodiments,and the skilled artisan should recognize that variations can be madewithin the spirit and scope of the invention as described in theforegoing specification.

1. A hydrohalide salt form of a polymer-active agent conjugatecorresponding to structure (I):

wherein each n is an integer ranging from about 20 to about 500 andgreater than 95 mole percent of irinotecan's basic nitrogen atoms areprotonated in hydrohalide (HX) salt form, where X is selected fromfluoride, chloride, bromide, and iodide.
 2. The hydrohalide salt ofclaim 1, wherein greater than 96 mole percent of irinotecan's basicnitrogen atoms are protonated in hydrohalide salt form.
 3. (canceled) 4.(canceled)
 5. The hydrohalide salt of claim 1, wherein the hydrohalidesalt is a hydrochloride salt.
 6. (canceled)
 7. The hydrochloride salt ofclaim 5, wherein n is an integer ranging from about 80 to about
 150. 8.(canceled)
 9. A method for preparing a composition comprising ahydrohalide salt of a polymer-active agent conjugate corresponding tostructure (I), the method comprising:

(i) treating glycine-irinotecan hydrohalide, where the amino group ofglycine is in protected form, with a molar excess of hydrohalic acid tothereby remove the protecting group to form deprotectedglycine-irinotecan hydrohalide, (ii) coupling the deprotectedglycine-irinotecan hydrohalide from step (i) with a polymer reagentbearing an active ester in the presence of a base to form4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt, and (iii)recovering the 4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt byprecipitation.
 10. The method of claim 9, where the recovered4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide possesses greaterthan 95 mole percent of irinotecan's basic nitrogen atoms in hydrohalide(HX) salt form.
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. Themethod of claim 9, further comprising (iv) analyzing the recovered4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt for halidecontent, and, in the event the halide content is less than 95 molepercent, (v) dissolving the recovered4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt in ethylacetate, and adding additional hydrohalic acid.
 15. (canceled)
 16. Themethod of claim 14, wherein the hydrohalic acid is added in the form ofan ethanol solution.
 17. The method of claim 16, wherein following theadding of additional hydrohalic acid in step (v), the4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan hydrohalide salt is recovered byprecipitation.
 18. (canceled)
 19. The method of claim 9, wherein theglycine-irinotecan hydrohalide in protected form is treated with aten-fold or greater molar excess of hydrohalic acid to thereby removethe protecting group to form glycine-irinotecan hydrohalide. 20.(canceled)
 21. The method of claim 9, wherein the glycine-irinotecanhydrohalide in protected form ister-butyloxycarbonyl(Boc)-glycine-irinotecan hydrochloride, where theamino group of glycine is Boc-protected.
 22. The method of claim 9,wherein the glycine-irinotecan hydrohalide in step (i) isglycine-irinotecan hydrochloride in protected form, and theglycine-irinotecan hydrochloride in protected form is treated withhydrochloric acid to remove the protecting group.
 23. (canceled)
 24. Themethod of claim 9, where step (i) further comprises isolating theglycine-irinotecan hydrohalide prior to step (ii).
 25. The method ofclaim 24, where the isolating of the glycine-irinotecan hydrohalideprior to step (ii) is by precipitation.
 26. (canceled)
 27. (canceled)28. (canceled)
 29. (canceled)
 30. The method of claim 9, where step (ii)is carried out in a chlorinated solvent.
 31. (canceled)
 32. Acomposition comprising a hydrohalide salt of4-arm-pentaerythritolyl-polyethyleneglycol-carboxymethyl-glycine-irinotecan prepared according to claim 9.33. A pharmaceutically acceptable composition comprising the hydrohalidesalt of claim 1 and a pharmaceutically acceptable excipient.
 34. Thepharmaceutically acceptable composition of claim 33 comprising lactatebuffer, in lyophilized form.
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. A method of treating cancer in a mammalian subject byadministering a therapeutically effective amount of a pharmaceuticallyacceptable composition of claim 33 to a subject diagnosed as having oneor more cancerous solid tumors over a duration of time effective toproduce an inhibition of solid tumor growth in the subject.
 39. Themethod of claim 38, wherein the cancerous solid tumor is selected fromthe group consisting of colorectal, ovarian, cervical, breast andnon-small cell lung.
 40. (canceled)
 41. The composition of claim 33,wherein the polymer reagent bearing an active ester is obtainable from amethod comprising: alkoxylating in a suitable solvent a previouslyisolated alkoxylatable oligomer to form an alkoxylated polymericmaterial, wherein the previously isolated alkoxylatable oligomer has aknown and defined weight-average molecular weight of greater than 300Daltons; modifying the alkoxylated polymeric material, in one or moresteps, to bear an active ester, thereby forming a polymer reagentbearing an active ester.
 42. The composition of claim 41, wherein thepolymer reagent bearing an active ester has the following structure:

wherein each n is from about 40 to about
 500. 43. A compositioncomprising hydrochloride salts of four-arm polymer conjugates, whereinat least 90% of the four-arm conjugates in the composition: (i) have astructure encompassed by the formula,C—[CH₂—O—(CH₂CH₂O)_(n)—CH₂—C(O)-Term]₄, wherein n, in each instance, isan integer having a value from 5 to 150 (e.g., about 113), and Term, ineach instance, is selected from the group consisting of —OH, —OCH₃,

—NH—CH₂—C(O)—OH, —NH—CH₂—C(O)—OCH₃,

and —NH—CH₂—C(O)—O-Irino (“GLY-Irino”), wherein Irino is a residue ofirinotecan; and (ii) for each Term in the at least 90% of the four-armconjugates in the composition, at least 90% thereof are—NH—CH₂—C(O)—O-Irino, and further wherein for each amino group withineach Irino in the at least 90% of the four-arm conjugates in thecomposition, each amino group will either be protonated or unprotonated,where any given protonated amino group is a hydrochloride salt.